Magnetic-nanoparticle Conjugates and Methods of Use

ABSTRACT

The present invention provides novel compositions of binding moiety-nanoparticle conjugates, aggregates of these conjugates, and novel methods of using these conjugates, and aggregates. The nanoparticles in these conjugates can be magnetic metal oxides, either monodisperse or polydisperse. Binding moieties can be, e.g., oligonucleotides, polypeptides, or polysaccharides. Oligonucleotide sequences are linked to either non-polymer surface functionalized metal oxides or with functionalized polymers associated with the metal oxides. The novel compositions can be used in assays for detecting target molecules, such as nucleic acids and proteins, in vitro or as magnetic resonance (MR) contrast agents to detect target molecules in living organisms.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/956,007, filed Dec. 1, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/256,804, filed Apr. 18, 2014, which is acontinuation of U.S. patent application Ser. No. 12/772,782, filed May3, 2010, which is a divisional of U.S. patent application Ser. No.12/194,475, filed on Aug. 19, 2008, which is a continuation of U.S.patent application Ser. No. 10/165,258, filed on Jun. 6, 2002, whichclaims priority to U.S. Provisional Patent Application Ser. No.60/296,378, filed on Jun. 6, 2001, the contents of each of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to magnetic nanoparticle conjugates and methodsof use.

BACKGROUND

Magnetic particles are widely used reagents for the purification andextraction of nucleic acids. For example, U.S. Pat. Nos. 4,554,488 and4,672,040 attach a nucleic acid to silanized magnetic particles that areabout one micron in diameter. U.S. Pat. No. 5,898,071 utilizesone-micron silanized particles in the presence of polyethylene glycol(PEG) to extract DNA. Similar magnetic particles have been used tocapture and release target nucleic acid for the detection of specificsequences of DNA (see, e.g., U.S. Pat. No. 5,750,338). Streptavidin hasbeen attached to magnetic particles and can be used to recoverbiotinylated nucleic acids after sequence specific hybridization.

U.S. Pat. No. 5,512,439 describes attaching oligonucleotides to 1-10micron microspheres impregnated with iron oxide and using themicrospheres to bind to target nucleic acids, which can then beseparated from their surrounding media.

U.S. Pat. No. 6,027,945 describes the use of magnetic silica particleshaving a diameter of 4-7 microns to extract and purify nucleic acidsincluding DNA fragments, plasmid DNA, and RNA.

U.S. Pat. No. 5,508,164 describes linking chromosomes to silanized glasssupports with disulfide crosslinking agents to link to cell organelles.Cell organelles are then labeled and biotinylated magnetic particles areused to magnetically recover the particles after separation from thesolid support using reducing agents.

Preparations of magnetic particles designed for separation andextraction use particles that are amenable to easy manipulation by weakapplied magnetic fields. These materials are typically micron sized andhave a high magnetic moment per particle; their effects on waterrelaxation rate are unspecified and not relevant to their application.Nanoparticles do not respond to the weak, magnetic fields of hand heldmagnets.

Magnetic particles have also been used to assay for analytes based ontheir ability to bind analytes and change magnetic resonance (MR)relaxation rates. In U.S. Pat. No. 5,164,297, bovine serum albumin (BSA)coated magnetic particles were used to react with an antibody. Additionof BSA favors dissociation of the complex between the antibody and theBSA-coated iron oxide particles. As a result of this dissociation ofaggregates, the solvent T2 relaxation time decreased (1/T2 increased)and the BSA concentration can be determined from T2.

In another example, WO 01/19405 describes the preparation and uses ofmagnetic nanoparticles with various biomacromolecules attached.

SUMMARY

The present invention provides new magnetic conjugates and methods fortheir synthesis and use. Each conjugate comprises a magneticnanoparticle linked to a binding moiety that specifically binds to atarget in a sample, such as a nucleic acid or protein, to anotherbinding moiety on another conjugate, or to an aggregation inducingmolecule, such as avidin. The novel compositions can be used in assaysfor detecting specific target molecules, such as biological molecules insample solutions, e.g., by altering the magnetic resonance (MR)relaxation rate of the solution. Thus, the new conjugates can beconsidered to be magnetic relaxation switches (MRS).

The biological target molecules can be nucleic acid sequences (e.g., asequence complementary to the binding moiety for hybridization), proteinsequences (e.g., an antibody binding site), or polysaccharide sequences.Biological molecules are molecules of biological origin or syntheticallymade molecules that mimic the performance of biological molecules.Examples include, but are not limited to, peptides with non-naturalamino acids, peptide nucleic acids (PNA's), or natural or man-madeorganic molecules that react with specific sites on target biologicalmolecules.

The assay method is accomplished by synthesizing a population and, insome aspects of the invention, at least two populations of the bindingmoiety-nanoparticle conjugates. Each conjugate in a population has aplurality, e.g., two, three, four, or more, of a single type of bindingmoiety attached to a nanoparticle. The nanoparticle is composed of amagnetic metal oxide and one or more functional groups, e.g., a polymercomprising one or more functional groups. When polymers are included,they contain functional groups that enable the binding moiety to beattached to the nanoparticle to form the conjugate. The polymer can be anatural polymer, a synthetic polymer, a combination of natural andsynthetic polymers, or derivatives of each type. The functional groupscan be carboxy, amino, or sulfhydryl groups. In some embodiments, thebinding moiety is attached to the nanoparticle through disulfide groups.The metal oxides can also be associated with non-polymer functionalgroups to form the nanoparticles.

In one aspect of the invention, a population of conjugates (or a mixtureof two or more populations of conjugates with differing binding moietiesdirected to the same target molecule or type of target molecule) isplaced into a sample solution. If the sample solution contains a targetmolecule to which the binding moieties specifically bind, the bindingmoieties interact with and bind to the target resulting in the formation(self-assembly) of aggregates, which are groups of 2 to about 20conjugates bound together via a target molecule, an aggregation inducingmolecule, or by their binding moieties. Thus, the dispersed state of theconjugates switches to an aggregate state, which decreases T2 relaxationtimes. FIG. 1 depicts one embodiment of such an interaction in which twoconjugates, P1 and P2, combine to form an aggregate of six conjugates.

In another aspect of the invention, small aggregates of conjugates areprepared and then placed into a sample solution. In this assay system,the binding moieties when bound together to form the aggregates form asubstrate that is cleaved by a specific target molecule, such as anenzyme that cleaves a specific site within a double-stranded nucleicacid formed by the hybridization of two single-strand oligonucleotidebinding moieties. Alternatively, the binding moieties can be bound to anaggregation inducing molecule to form the aggregate. If the samplesolution contains a target molecule, the substrate formed by the bindingmoieties is cleaved, resulting in the dissolution of the aggregates.Thus, the aggregate state switches to a dispersed state, which increasesT2 relaxation times.

In various specific embodiments, the magnetic metal oxide containssuperparamagnetic iron oxide crystals. The superparamagnetic characterof the iron oxide of the nanoparticle makes it a potent enhancer ofwater relaxation rates, an enhancement that is altered when a targetmolecule binds specifically with a binding moiety of a conjugate tocreate an aggregate. Aggregates exert sensitive and reversible effectson the spin-spin relaxation of adjacent water protons upon hybridizationin fluid phase. As a consequence, the presence of the target moleculeinteracting with the conjugate, in one embodiment, decreases T2.

The assays can be run in a single tube or in an array format, i.e., T2can be determined for a single sample or for a number of samplessimultaneously. The assays can be performed in fluid media that areoptically transparent, optically translucent, optically opaque, or inturbid solutions. The fluid can be water, saline, buffered saline, orbiological fluids, such as, but not limited to, blood, cell and/ortissue homogenates, extracts, suspensions, saliva, semen, milk, spinalfluid, and urine. Target molecules include sequences of nucleic acidsunique to specific microorganisms such as viruses or bacteria or mRNA ofspecific genes expressed in specific cells (see below). The methods canalso be used for genotyping DNA from humans or mammals.

In one embodiment, the invention features an aggregate including aplurality of conjugates, wherein each conjugate includes a magneticnanoparticle linked to a binding moiety that specifically binds to atarget molecule, to another binding moiety, or to an aggregationinducing molecule, and wherein each conjugate within the aggregate isbound to at least one other conjugate in the aggregate through theirrespective binding moieties. The aggregates can include 2 to about 20conjugates, and can have a size of about 100 to 500 nm, e.g., 200, 300,or 400 nm.

The aggregates can also include a target molecule to which at least twodifferent binding moieties specifically bind. In certain embodiments,the target molecule is a nucleic acid, and each binding moiety includesone of two or more different oligonucleotides, wherein eacholigonucleotide is complementary to a region on the target nucleic acidthat is different than the regions to which the other oligonucleotidesare complementary. In other embodiments, the target molecule can be apolypeptide, and each binding moiety includes one of two or moredifferent antibodies, wherein each antibody specifically binds to abinding site on the polypeptide that is different than the binding sitesto which the other antibodies bind.

The aggregates can further include an aggregation inducing molecule,such as avidin or an antibody, wherein the binding moieties eachselectively bind to the aggregation inducing molecule. For example, thebinding moieties can include biotin.

In some embodiments, the binding moieties bind to each other to form theaggregate. For example, each binding moiety can include a cleavage sitethat is selectively cleaved by a target molecule, and cleavage of thebinding moiety results in separation of the conjugates and dispersal ofthe aggregate. Alternatively, the binding moieties can be polypeptidesand the target molecule can be an enzyme. In other examples, eachbinding moiety can bind to another binding moiety to form a cleavagesite that is selectively cleaved by a target molecule, and cleavage ofthe binding moiety results in dispersal of the aggregate. For example,each binding moiety can include one of two complementary single-strandedoligonucleotides that hybridize to form a double-stranded nucleic acidcomprising a cleavage site, and wherein the target molecule is anendonuclease.

“Linked” means attached or bound by covalent bonds, or non-covalentbonds, or other bonds, such as van der Waals forces.

“Specifically binds” means that one molecule, such as a binding moiety,e.g., an oligonucleotide or antibody, binds preferentially to anothermolecule, such as a target molecule, e.g., a nucleic acid or a protein,in the presence of other molecules in a sample.

In another aspect, the invention features a composition including amixture of at least two populations of conjugates that specifically bindto a target molecule, wherein each conjugate in the first populationcomprises a nanoparticle including a magnetic metal oxide (e.g., asuperparamagnetic metal oxide) linked to a plurality (e.g., two orthree) of first binding moieties (e.g., oligonucleotides, polypeptidessuch as antibodies, and polysaccharides) that bind to a first bindingsite on the target molecule, and wherein each conjugate in the secondpopulation comprises a nanoparticle comprising a magnetic metal oxidelinked to a plurality of second binding moieties that bind to a secondbinding site on the target molecule.

These compositions can include conjugates that further includefunctional groups that link the nanoparticles to the binding moieties.The functional groups can be amino, carboxy, or sulfhydryl groups.Alternatively, the conjugates can further include a polymer associatedwith the nanoparticles, and wherein the functional groups are bound tothe polymer and to the binding moieties. The polymers can behydrophilic, a natural or synthetic polymer, or a derivative of anatural or synthetic polymer. Examples of polymers include dextran,carboxymethyl dextran, reduced carboxymethyl dextran, crosslinkedaminated dextran, pullanan, polyethylene glycol, and silane. In someembodiments, the binding moieties are attached to the functional groupsthrough a covalent bond or by a disulfide bond. For example,oligonucleotides can be attached to the nanoparticles by a singlecovalent bond at the 3′ or 5′ end of each oligonucleotide.

In these compositions, the magnetic metal oxide can have a diameterbetween about 1 nm and about 25 nm, and the conjugate can have adiameter between about 15 nm and 100 nm, e.g., between about 40 nm andabout 60 nm. In addition, each conjugate in the composition can have anR1 relaxivity between about 5 and 30 mM⁻¹ sec⁻¹ and an R2 relaxivitybetween about 15 and 100 mM⁻¹ sec⁻¹. In particular embodiments, thenanoparticle is an amino-derivatized cross-linked iron oxidenanoparticle.

In another aspect, the invention features a conjugate including amagnetic nanoparticle linked to a first binding moiety, wherein thefirst binding moiety includes a cleavage site for a target molecule andspecifically binds to an aggregation inducing molecule, forms a cleavagesite for the target molecule when the first binding moiety binds to asecond binding moiety, or specifically binds to an aggregation inducingmolecule that comprises a cleavage site. For example, the first bindingmoiety can include a polypeptide that has the cleavage site, and thetarget molecule can be an enzyme. Alternatively, the first bindingmoiety can bind to the second binding moiety to form the cleavage sitethat is selectively cleaved by a target molecule, wherein the targetmolecule is an enzyme. For example, the first and second bindingmoieties can be complementary single-stranded oligonucleotides thathybridize to form a double-stranded nucleic acid comprising the cleavagesite, wherein the target molecule is an endonuclease.

In another example, the first binding moiety includes a polypeptide thatcontains the cleavage site and biotin, and the aggregation inducingmolecule is avidin, or the first binding moiety includes avidin and theaggregation inducing molecule includes biotin and the cleavage site. Theaggregation inducing molecule can also be an oligonucleotide with abiotin molecule at each end, wherein the cleavage site is an internalsite, e.g., a site that is not at either end of the oligonucleotide. Theaggregation inducing molecule can also include a polypeptide with abiotin molecule at each end, where the cleavage site is an internalsite.

In another aspect, the invention features a method for determining thepresence of a target molecule in a sample, by obtaining a mixture of atleast two populations of conjugates that specifically bind to the targetmolecule to form an aggregate, wherein each conjugate in the firstpopulation includes a nanoparticle that includes a magnetic metal oxidelinked to a plurality of first binding moieties that bind to a firstbinding site on the target molecule, and wherein each conjugate in thesecond population includes a nanoparticle including a magnetic metaloxide linked to a plurality of second binding moieties that bind to asecond binding site on the target molecule; contacting the mixture witha fluid sample under conditions that enable the first and second bindingmoieties to specifically bind to any target molecules in the sample andform an aggregate of conjugates; and determining the presence of anaggregate in the sample, wherein the presence of the aggregate indicatesthe presence of the target molecule.

In this method, the presence of an aggregate can be determined byobtaining the relaxation properties of the fluid in the sample, whereina change in the relaxation properties of the fluid indicates thepresence of the target molecule. For example, a decrease in spin-spinrelaxation time (T2) indicates the presence of the target molecule.

In certain embodiments, the target molecule is a nucleic acid, the firstbinding moieties are first oligonucleotides that are complementary to afirst region of the target nucleic acid, and the second binding moietiesare second oligonucleotides that are complementary to a second region ofthe target nucleic acid. In other embodiments, the target molecule is apolypeptide, the first binding moieties are first antibodies thatspecifically bind to a first binding site of the target polypeptide, andthe second binding moieties are second antibodies that specifically bindto a second binding site of the target polypeptide. For example, thefirst and second antibodies can be monoclonal antibodies.

In another aspect, the invention features a method for determining thepresence of a target molecule in a sample, by obtaining one or morepopulations of conjugates that are capable of forming an aggregate,wherein each conjugate in a first population includes a nanoparticleincluding a magnetic metal oxide linked to a first binding moiety,wherein the first binding moiety includes a cleavage site for the targetmolecule and specifically binds to an aggregation inducing molecule,forms a cleavage site for the target molecule when the first bindingmoiety binds to a second binding moiety in a second population ofconjugates, or specifically binds to an aggregation inducing moleculethat includes a cleavage site; mixing the conjugates of the one or morepopulations in a fluid under conditions that enable the binding moietiesto specifically bind to each other or to an aggregation inducingmolecule to form aggregates in the fluid; mixing the fluid containingthe aggregates with a fluid sample under conditions that enable anytarget molecules in the sample to cleave the cleavage sites in theaggregates; and determining the presence of aggregates in the sample,wherein the absence of aggregates indicates the presence of the targetmolecule.

In this method, the absence of aggregates can be determined by obtainingthe relaxation properties of the fluid in the sample, wherein a changein the relaxation properties of the fluid indicates the presence of thetarget molecule. For example, an increase in spin-spin relaxation time(T2) indicates the presence of the target molecule.

In this method, the target molecule can be an enzyme, and the firstbinding moiety can include a polypeptide that contains the cleavagesite. In addition, the binding moieties can be a polypeptide thatcontains the cleavage site and biotin, and the aggregation inducingmolecule can be avidin. In some embodiments, the target molecule is anendonuclease, and the first and second binding moieties arecomplementary single-stranded oligonucleotides that hybridize to form adouble-stranded nucleic acid comprising the cleavage site selectivelycleaved by the endonuclease. In other embodiments, the first bindingmoiety includes avidin and the aggregation inducing molecule includesbiotin and the cleavage site. For example, the aggregation inducingmolecule can include an oligonucleotide or polypeptide with a biotinmolecule at each end, and the cleavage site is an internal site.

In another aspect, the invention features a method for determining thepresence of a target molecule in a sample, by obtaining first and secondpopulations of oligonucleotide-nanoparticle conjugates, wherein eachconjugate in the first population includes a nanoparticle having amagnetic metal oxide associated with a polymer having functional groups;and a plurality of first oligonucleotides attached to the functionalgroups on the nanoparticle; and wherein each conjugate in the secondpopulation includes a nanoparticle having a metal oxide associated witha polymer having functional groups; and a plurality of secondoligonucleotides attached to the functional groups on the nanoparticle;wherein the first and second oligonucleotides are each complementary tofirst and second portions of the target nucleic acid, and wherein theoligonucleotides in each population are the same on each conjugate inthe population and different than the oligonucleotides on the conjugatesin the other populations; preparing a mixture of the first and secondpopulations of oligonucleotide-nanoparticle conjugates; obtaining afluid sample; contacting the mixture with the sample under conditionsthat enable any target nucleic acid in the sample to hybridize to thefirst and second oligonucleotides of both populations of conjugates; andobtaining the relaxation properties of the fluid in the sample, whereina change in the relaxation properties of the fluid indicates thepresence of the target nucleic acid.

In other aspects, the invention includes assays for determiningaggregate formation in a fluid sample by adding a new composition of theinvention to the fluid sample under conditions that enable aggregateformation; and measuring the relaxation properties of the sample overtime after addition of the composition, wherein a decrease in spin-spinrelaxation (T2) indicates aggregate formation. In another version, theinvention includes assays for determining aggregate dispersal in a fluidsample by adding an aggregate formed from the new conjugates to thefluid sample under conditions that enable cleavage of the aggregate;measuring the relaxation properties of the sample over time afteraddition of the conjugate, wherein an increase in spin-spin relaxation(T2) indicates aggregate dispersal.

In the new methods, the sample fluid can be optically transparent,optically translucent, optically turbid, or optically opaque. The fluidcan be water, saline, buffered saline, or a biological fluid. Thebiological fluid can be blood, a cell homogenate, a tissue homogenate, acell extract, a tissue extract, a cell suspension, a tissue suspension,milk, urine, saliva, semen, or spinal fluid.

In these methods, the amount of change in the relaxation properties canindicate a concentration of target molecules in the sample.

In another aspect, the invention features a method for purifying atarget molecule (such as a nucleic acid or polypeptide) from a sample byobtaining a conjugate including a nanoparticle having a magnetic metaloxide linked by a cleavable bond (e.g., a reducible disulfide bond) to abinding moiety that specifically binds to a binding site on the targetmolecule; obtaining a sample containing the target molecule in a fluid;mixing the conjugates with the sample under conditions sufficient toenable target molecules in the sample to bind to the binding moiety onthe conjugate to form target molecule-binding moiety complexes;separating the conjugates from the sample; and cleaving the cleavablebond to separate the target molecule-binding moiety complexes from theconjugates, thereby purifying the target molecules.

In the methods for the extraction and purification of nucleic acids ormaterials hybridizing to nucleic acids. The conjugates can beoligonucleotide-nanoparticle conjugates having a reducible disulfidebond to couple the oligonucleotides to the nanoparticles, and as aresult reducing agents can separate the oligonucleotides from thenanoparticles at a desired time. Materials bound to the oligonucleotideportion of these oligonucleotide nanoparticle conjugates, such asdouble-stranded nucleic acids, can be obtained by the use of reducingagents rather than by use of the high ionic strength, chaotropic agents,or extremes of pH, that are required in previously known methods.

The invention also includes an assay for simultaneously determining thepresence of a target nucleic acid in a plurality of samples, byobtaining first and second populations of oligonucleotide-nanoparticleconjugates, wherein each conjugate in the first population includes ananoparticle having a magnetic metal oxide associated with a polymerhaving functional groups; and a plurality of first oligonucleotidesattached to the functional groups on the nanoparticle, and wherein eachconjugate in the second population includes a nanoparticle having ametal oxide associated with a polymer having functional groups; and aplurality of second oligonucleotides attached to the functional groupson the nanoparticle; wherein the first and second oligonucleotides areeach complementary to first and second portions of the target nucleicacid, and wherein the oligonucleotides in each population are the sameon each conjugate in the population and different than theoligonucleotides on the conjugates in the other populations; preparing amixture of the first and second populations ofoligonucleotide-nanoparticle conjugates; obtaining a plurality of fluidsamples; contacting a portion of the mixture with each of the pluralityof samples under conditions that enable any target nucleic acid in thesamples to hybridize to the first and second oligonucleotides of bothpopulations of oligonucleotide-nanoparticle conjugates; andsimultaneously obtaining the relaxation properties of the fluid in eachof the plurality of samples, wherein a change in the relaxationproperties of a sample indicates the presence of the target nucleic acidin that sample.

In this method, the amount of change in the relaxation properties of asample can indicate a concentration of target nucleic acid in thatsample.

The invention also features a method for determining the presence of atarget molecule in a subject by administering to the subject at leastone population of conjugates, wherein each conjugate includes ananoparticle having a magnetic metal oxide linked to a binding moietythat specifically binds to the target molecule; providing sufficienttime for the binding moieties to bind to target molecules in thesubject; and generating a magnetic resonance (MR) image of the subject,wherein a signal in the image indicates the presence of a targetmolecule. For example, the target molecule can be a nucleic acid orprotein, and the binding moiety can be an oligonucleotide that iscomplementary or an antibody that specifically binds to a portion of thetarget nucleic acid. In one embodiment, at least two populations ofconjugates are administered to the subject, wherein the binding moietieson the conjugates in each population are identical within the populationand different from the binding moieties on the conjugates in otherpopulations; and wherein the binding moieties in different populationsof conjugates specifically bind to different portions of the targetmolecule.

In another aspect, the invention features a method for determining thelevels of mRNA in cells using a mixture of populations ofsuperparamagnetic oligonucleotide-iron oxide nanoparticle conjugates andMR imaging. When the conjugates react with a target, e.g., mRNA, therelaxation rate of the solvent changes. Hence, the level of an mRNA in asample can be determined from measurements of spin-spin or spin-latticerelaxation times of water. In another embodiment, a detector is usedthat can simultaneously measure the relaxation properties of manyspatially separated samples simultaneously, e.g., in an array format. Inyet another embodiment, the invention features a method in which theoligonucleotide-nanoparticle conjugates and an MR detector are used todetermine the pattern of gene expression in an organism.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing a reaction scheme in whichalkanethiooligonucleotides were reacted with N-succinimidyl3-(2-pyridyldithio) propionate (SPDP) activated nanoparticles to formnanoparticle conjugates P1 and P2. P1 and P2 hybridize withcomplementary oligonucleotides followed by aggregation and magneticrelaxivity changes. Dithiothreitol (DTT) treatment breaks the bondbetween nanoparticle and alkanethiooligonucleotide.

FIGS. 2A to 2D are images of test tubes illustrating the effect ofincubating oligonucleotide-nanoparticle conjugates witholigonucleotides. From left to right, 2A: P1 and P2; 2B: P1, P2 pluscomplementary oligonucleotide; 2C: P1, P2 plus half-complementaryoligonucleotide; 2D: P1, P2 and non-complementary oligonucleotide. Theprecipitate in the tube in FIG. 2B was moved to the side with a handheld magnet as indicated by two arrows.

FIGS. 3A and 3B are images of gel electrophoresis of aP1/P2/oligonucleotide nanoparticle precipitate. FIG. 3A shows a gel runin non-denaturing conditions. Lane 1: No DTT. Oligonucleotide remainswith P1/P2 at the top of the gel (arrow). Lane 2: With DTT. A singleband of double stranded oligonucleotide is seen. FIG. 3B shows a gel runwith denaturing conditions and with DTT. Two bands are evident, thecomplementary oligonucleotide (slower band) and a band of 5′ and 3′alkanethioligonucleotides (faster band).

FIG. 4 is a graph illustrating the temporal change of water T2relaxation times with (square) and without complementary oligonucleotide(diamond). The insert shows the effect of increasing concentrations ofcomplementary oligonucleotide on T2. All data points shown represent theaverage of three measurements with standard deviations ranging between0.4-0.6 msec for T2 values (too small to graph).

FIG. 5 is a graph illustrating the T2 changes of an aqueous solution ofP1/P2/complementary oligonucleotide as a function of temperaturecycling. T2 is different between two solutions of P1/P2, one withcomplementary and without complementary oligonucleotide. At 80° C. thereis no hybridization and a very small difference in T2 values.

FIG. 6 is a graph showing T2 values of a turbid medium (INTRALIPID®)after a complementary oligonucleotide is added to an oligonucleotidenanoparticle conjugate mixture, P1 and P2. DTT was added after 180minutes.

FIG. 7 is an MR image showing the signal intensity of 24 wells of a 384well microtiter plate. Wells had 3 or 6 Tg Fe/mL as mixture of P1 andP2. Wells had the indicated amounts of either complementary ornoncomplementary oligonucleotide.

FIG. 8A is a graph depicting the specificity of magnetic nanosensors.Temporal change of T2 relaxation times of P1-GFP and P2-GFP with theaddition of various target oligonucleotides containing single nucleotidemismatches G, T, C. The perfect target sequence is clearly distinguishedfrom single nucleotide mismatches. FIG. 8B is an MR image correspondingto the graph in FIG. 8A acquired 2 hours after hybridization indicatingthat T2 relaxation time measurements correlate with the fluorescencemeasurements

FIG. 9A is an image of a section of a 384 well plate containing GFP-P1and GFP-P2 with total RNA extracted from various cell lines. FIG. 9B isan image of the nanoparticle conjugates with lysed cells from WT orGFP+human glioma lysate two hours following hybridization. FIG. 9C is agraph illustrating GFP fluorescence and T2 relaxation time measurementsof GFP mRNA indicating that the two measurements correlate well in wholecell lysate experiments indicating that mRNA is readily detectable by MRimaging.

FIG. 10A is a graph illustrating the incubation of anti-GFP-P1nanoparticle conjugates with GFP or BSA protein resulting in asignificant decrease in T2. FIG. 10B is a graph illustrating theincubation of an aggregate of conjugates, each containing a DEVDpeptide, with the enzyme caspase, which cleaves the DVED peptidesequence resulting in a dissolution of the conjugates, and an increaseof T2 relaxation time.

FIG. 11 is a schematic image of a small aggregate held together bydouble stranded oligonucleotide (P1/P2). Sequence specific cleavage byBamHI results in separation of the particles in the aggregate with acorresponding increase in T2 relaxation times.

FIGS. 12A and 12B are atomic force micrographs, of the conjugates ofFIG. 11 before (aggregates of FIG. 12A) and after (dispersed conjugatesof FIG. 12B) addition of BamHI treatment.

DETAILED DESCRIPTION

The present invention provides compositions of conjugates and aggregatesof conjugates, and methods of making and using these conjugates andaggregates. Each conjugate comprises one or more binding moieties (e.g.,an oligonucleotide, nucleic acid, polypeptide, or polysaccharide)linked, e.g., covalently or non-covalently, to a magnetic, e.g.,superparamagnetic, nanoparticle. The binding moiety causes a specificinteraction with a target molecule (or, in some embodiments, anaggregation inducing molecule, such as avidin). Either, the bindingmoiety specifically binds to a selected target molecule, which can be,for example, a nucleic acid, polypeptide, or polysaccharide, or thebinding moiety can be designed to bind to another binding moiety to forma substrate that is cleaved by the target molecule. Binding causesaggregation of the conjugates, resulting in a decrease of the spin-spinrelaxation time (T2) of adjacent water protons in an aqueous solution.Cleavage causes dispersal of the aggregate into separate conjugates,resulting in an increase of the spin-spin relaxation time (T2) ofadjacent water protons in an aqueous solution.

Nanoparticles

Nanoparticles can be monodisperse (a single crystal of a magneticmaterial, e.g., metal oxide, such as superparamagnetic iron oxide, pernanoparticle) or polydisperse (a plurality of crystals, e.g., 2, 3, or4, per nanoparticle). The magnetic metal oxide can also comprise cobalt,magnesium, zinc, or mixtures of these metals with iron. The term“magnetic” as used in this specification and the accompanying claimsmeans materials of high positive magnetic susceptibility such assuperparamagnetic compounds and magnetite, gamma ferric oxide, ormetallic iron. Important features and elements of nanoparticles that areuseful to produce the new conjugates include: (i) a high relaxivity,i.e., strong effect on water relaxation, (ii) a functional group towhich the binding moiety can be covalently attached, (iii) a lownon-specific binding of interactive moieties to the nanoparticle, and(iv) stability in solution, i.e., the nanoparticles do not precipitate.

In all embodiments, the nanoparticles are attached (linked) to thebinding moieties via functional groups. In some embodiments, thenanoparticles are associated with a polymer that includes the functionalgroups, and also serves to keep the metal oxides dispersed from eachother. The polymer can be a synthetic polymer, such as, but not limitedto, polyethylene glycol or silane, natural polymers, or derivatives ofeither synthetic or natural polymers or a combination of these. Usefulpolymers are hydrophilic. In some embodiments, the polymer “coating” isnot a continuous film around the magnetic metal oxide, but is a “mesh”or “cloud” of extended polymer chains attached to and surrounding themetal oxide. The polymer can comprise polysaccharides and derivatives,including dextran, pullanan, carboxydextran, carboxmethyl dextran,and/or reduced carboxymethyl dextran. The metal oxide can be acollection of one or more crystals that contact each other, or that areindividually entrapped or surrounded by the polymer.

In other embodiments, the nanoparticles are associated withnon-polymeric functional group compositions. Methods are known tosynthesize stabilized, functionalized nanoparticles without associatedpolymers, which are also within the scope of this invention. Suchmethods are described, for example, in Halbreich et al., Biochimie, 80(5-6):379-90, 1998.

The nanoparticles have an overall size of less than about 1-100 nm. Themetal oxides are crystals of about 1-25 nm, e.g., about 3-10 nm, orabout 5 nm in diameter. The polymer component in some embodiments can bein the form of a coating, e.g., about 5 to 20 nm thick or more. Theoverall size of the nanoparticles is about 15 to 200 nm, e.g., about 20to 100 nm, about 40 to 60 nm; or about 50 nm.

The conjugates have high relaxivity owing to the superparamagnetism oftheir iron or metal oxide. They have an R1 relaxivity between about 5and 30 mM⁻¹ sec⁻¹, e.g., 10, 15, 20, or 25 mM⁻¹ sec⁻¹. They have an R2relaxivity between about 15 and 100 mM⁻¹ sec⁻¹, e.g., 25, 50, 75, or 90mM⁻¹ sec⁻¹. They typically have a ratio of R2 to R1 of between 1.5 and4, e.g., 2, 2.5, or 3. They typically have an iron oxide content that isgreater than about 10% of the total mass of the particle, e.g., greaterthan 15, 20, 25 or 30 percent.

Synthesis of Nanoparticles

There are varieties of ways that the nanoparticles can be prepared, butin all methods, the result must be a nanoparticle with functional groupsthat can be used to link the nanoparticle to the binding moiety.

For example, oligonucleotide binding moieties can be linked to the metaloxide through covalent attachment to a functionalized polymer or tonon-polymeric surface-functionalized metal oxides. In the latter method,the nanoparticles can be synthesized according to the method of Albrechtet al., Biochimie, 80 (5-6): 379-90, 1998. Dimercapto-succinic acid iscoupled to the iron oxide and provides a carboxyl functional group. Byfunctionalized is meant the presence of amino or carboxyl or otherreactive groups (see, Table 1, which is described in further detailbelow).

In another embodiment, oligonucleotides are attached to magneticnanoparticles via a functionalized polymer associated with the metaloxide. In some embodiments, the polymer is hydrophilic. In a specificembodiment, the conjugates are made using oligonucleotides that haveterminal amino, sulfhydryl, or phosphate groups, and superparamagneticiron oxide nanoparticles bearing amino or carboxy groups on ahydrophilic polymer. There are several methods for synthesizing carboxyand amino derivatized-nanoparticles. Methods for synthesizingfunctionalized, coated nanoparticles are discussed in further detailbelow.

Carboxy functionalized nanoparticles can be made, for example, accordingto the method of Gorman (see WO 00/61191). In this method, reducedcarboxymethyl (CM) dextran is synthesized from commercial dextran. TheCM-dextran and iron salts are mixed together and are then neutralizedwith ammonium hydroxide. The resulting carboxy functionalizednanoparticles can be used for coupling amino functionalizedoligonucleotides, see Table 1.

Carboxy-functionalized nanoparticles can also be made frompolysaccharide coated nanoparticles by reaction with bromo orchloroacetic acid in strong base to attach carboxyl groups. In addition,carboxy-functionalized particles can be made from amino-functionalizednanoparticles by converting amino to carboxy groups by the use ofreagents such as succinic anhydride or maleic anhydride.

Nanoparticle size can be controlled by adjusting reaction conditions,for example, by using low temperature during the neutralization of ironsalts with a base as described in U.S. Pat. No. 5,262,176. Uniformparticle size materials can also be made by fractionating the particlesusing centrifugation, ultrafiltration, or gel filtration, as described,for example in U.S. Pat. No. 5,492,814.

Nanoparticles can also be synthesized according to the method of Molday(Molday, R. S. and D. MacKenzie, “Immunospecific ferromagneticiron-dextran reagents for the labeling and magnetic separation ofcells,” J. Immunol. Methods, 1982, 52(3):353-67, and treated withperiodate to form aldehyde groups. The aldehyde-containing nanoparticlescan then be reacted with a diamine (e.g., ethylene diamine orhexanediamine), which will form a Schiff base, followed by reductionwith sodium borohydride or sodium cyanoborohydride.

Dextran-coated nanoparticles can be made and cross-linked withepichlorohydrin. The addition of ammonia will react with epoxy groups togenerate amine groups, see Hogemann, D., et al., Improvement of MRIprobes to allow efficient detection of gene expression Bioconjug. Chem.2000. 11(6):941-6, and Josephson et al., “High-efficiency intracellularmagnetic labeling with novel superparamagnetic-Tat peptide conjugates,”Bioconjug. Chem., 1999, 10(2):186-91. This material is known ascross-linked iron oxide or “CLIO” and when functionalized with amine isreferred to as amine-CLIO or NH₂-CLIO.

Carboxy-functionalized nanoparticles can be converted toamino-functionalized magnetic particles by the use of water-solublecarbodiimides and diamines such as ethylene diamine or hexane diamine.

Avidin or streptavidin can be attached to nanoparticles for use with abiotinylated binding moiety, such as an oligonucleotide or polypeptide.See e.g., Shen et al., “Magnetically labeled secretin retains receptoraffinity to pancreas acinar cells,” Bioconjug. Chem., 1996, 7(3):311-6.Similarly, biotin can be attached to a nanoparticle for use with anavidin-labeled binding moiety.

In all of these methods, low molecular weight compounds can be separatedfrom the nanoparticles by ultra-filtration, dialysis, magneticseparation, or other means. The unreacted oligonucleotides can beseparated from the oligonucleotide-nanoparticle conjugates, e.g., bymagnetic separation or size exclusion chromatography.

Binding Moieties

The binding moiety is a molecule, synthetic or natural, thatspecifically binds to, e.g., covalently or non-covalently binds to orhybridizes with, a target molecule, or with another binding moiety (or,in certain embodiments, with an aggregation inducing molecule). Forexample, the binding moiety can be a synthetic oligonucleotide thathybridizes to a specific complementary nucleic acid target. The bindingmoiety can also be an antibody directed toward an antigen or anyprotein-protein interaction. Also, the binding moiety can be apolysaccharide that binds to a corresponding target. In certainembodiments, the binding moieties can be designed or selected to serve,when bound to another binding moiety, as substrates for a targetmolecule such as enzyme in solution.

Oligonucleotide Binding Moieties

In certain embodiments, the binding moieties are oligonucleotides,attached to the nanoparticles using any one of a variety of chemistries,by a single, e.g., covalent, bond, e.g., at the 3′ or 5′ end to afunctional group on the nanoparticle.

The new conjugates are useful in various types of MR applications,including but not limited to, in vitro methods for assaying the presenceor concentration of nucleic acids, and in vivo methods as MR imagingagents.

An oligonucleotide binding moiety of the invention can be constructedusing chemical synthesis. A double-stranded DNA binding moiety can beconstructed by enzymatic ligation reactions using procedures known inthe art. For example, a nucleic acid (e.g., an oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the complementary strands, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Thenucleic acid also can be produced biologically using an expressionvector into which a nucleic acid has been subcloned.

One of the new in vitro assay methods uses at least two populations ofoligonucleotide magnetic nanoparticles, each with strong effects onwater relaxation (see Table 2). As the oligonucleotide-nanoparticleconjugates react with a target oligonucleotide, they form aggregates(100-500 nm; aggregates were 215 nm in size in Table 2). Upon prolongedstanding, e.g., overnight at room temperature, the aggregates form largeclusters (micron-sized particles), which settle out of solution, seeFIG. 2B. The invention uses magnetic resonance to determine therelaxation properties of the solvent, which are altered when the mixtureof magnetic oligonucleotide nanoparticles reacts with a target nucleicacid to form aggregates.

A feature of the analytical method when using oligonucleotide bindingmoieties is the need for a mixture of at least two types of magneticmetal oxide nanoparticles, each with a specific sequence ofoligonucleotide, and each with more than one copy of the oligonucleotideattached, e.g., covalently, per nanoparticle. The assay protocolinvolves preparing a mixture of populations ofoligonucleotide-nanoparticle conjugates and reacting the mixture with atarget nucleic acid. Alternatively, oligonucleotide-nanoparticleconjugates can be reacted with the target in a sequential fashion. Asecond feature of the new analytical method is the use of magneticresonance to detect the reaction of the oligonucleotide-nanoparticleconjugates with the target nucleic acid. When a target is present, thedispersed conjugates self-assemble to form small aggregates.

Synthesis of Oligonucleotides

The oligonucleotides used to make the conjugates are preferablydeoxyribonucleotides. Ribose-based oligonucleotides can also be useused, provided care is taken to eliminate RNA digesting enzymes. Theoligonucleotide can be synthesized with a single reactive group at the3′ or 5′ end, as indicated in Table 1, by methods known in the art. Thereactive group at the 3′ or 5′ end insures covalent attachment on oneend of the oligonucleotide. These can have 3′ or 5′ amino, phosphate, orsulfhydryl groups. One useful method, as described in Example 2,includes the use of 5′-alkanethiol-oligonucleotide and3′-alkanethiol-oligonucleotides. Oligonucleotides can be obtained fromnumerous commercial sources. Alternatively, oligonucleotides with biotinattached at the 3′ or 5′ end can be synthesized by methods known in theart, and used in conjunction with an avidin-bound nanoparticle.

Polypeptide Binding Moieties

In certain embodiments, the binding moiety is a polypeptide (i.e., aprotein, polypeptide, or peptide), attached, using any of a variety ofchemistries, by a single covalent bond in such a manner so as to notaffect the biological activity of the polypeptide. In one embodiment,attachment is done through the thiol group of single reactive cysteineresidue so placed that its modification does not affect the biologicalactivity of the polypeptide. In this regard the use of linearpolypeptides, with cysteine at the C-terminal or N-terminal end,provides a single thiol in a manner similar to which alkanethiolsupplies a thiol group at the 3′ or 5′ end of an oligonucleotide.Similar bifunctional conjugation reagents, such as SPDP and reactingwith the amino group of the nanoparticle and thiol group of thepolypeptide, can be used with any thiol bearing binding moiety. Thetypes of polypeptides used as binding moieties can be antibodies,antibody fragments, and natural and synthetic polypeptide sequences. Inall embodiments, these peptide binding moieties must have a bindingpartner, a molecule to which they selectively bind.

Use of peptides as binding moieties offers several advantages: First,the mass per binding site is low. For example, up to twenty 2 kDapeptides can be attached to a nanoparticle, calculated assuming 2064iron atoms per nanoparticle. With larger binding moieties like proteins(generally greater than about 30 kDa) the same mass of attachedpolypeptide results in only approximately 1-4 binding moieties pernanoparticle. Second, polypeptides can be engineered to have uniquelyreactive residues, distal from the residues required for biologicalactivity, for attachment to the nanoparticle. The reactive residue canbe a cysteine thiol, an N-terminal amino group, a C-terminal carboxylgroup or a carboxyl group of aspartate or glutamate, etc. A singlereactive residue on the peptide is used to insure a unique site ofattachment. These design principles can be followed with chemicallysynthesized peptides or biologically produced polypeptides.

The binding moieties can also contain amino acid sequences fromnaturally occurring (wild-type) polypeptides or proteins. For example,the natural polypeptide may be a hormone, (e.g., a cytokine, a growthfactor), a serum protein, a viral protein (e.g., hemagglutinin), anextracellular matrix protein, a lectin, or an ectodomain of a cellsurface protein. In each case, the resulting binding moiety-nanoparticleis used to measure the presence of analytes in a test media reactingwith the binding moiety.

Examples of protein hormones include: platelet-derived growth factor(PDGF) which binds the PDGF receptor; insulin-like growth factor-I and-II (Igf) which binds the Igf receptor; nerve growth factor (NGF) whichbinds the NGF receptor; fibroblast growth factor (FGF) which binds theFGF receptor (e.g., aFGF and bFGF); epidermal growth factor (EGF) whichbinds the EGF receptor; transforming growth factor (TGF, e.g., TGF-α andTGF-β) which bind the TGF receptor; erythropoietin, which binds theerythropoitin receptor; growth hormone (e.g., human growth hormone)which binds the growth hormone receptor; and proinsulin, insulin,A-chain insulin, and B-chain insulin, which all bind to the insulinreceptor.

Receptor binding moieties are useful for detecting and imaging receptorclustering on the surface of a cell.

Useful ectodomains include those of the Notch protein, Delta protein,integrins, cadherins, and other cell adhesion molecules.

Polypeptide Synthesis Methods for synthesizing polypeptides in solutionare well established in the field.

Solid-phase peptide synthesis (SPPS) can be used effectively to producepeptides and small proteins of specific sequences for use in the presentinvention.

The concept of the solid-phase approach involves covalent attachment(anchoring) of the growing peptide chain to an insoluble polymericsupport (resin carrier), so that unreacted soluble reagents can beremoved by simple filtration and washing without manipulative losses.Subsequently, the insoluble peptide-resin is extended by a series ofadditional cycles, which are required to proceed with high yields andfidelities. Excess soluble reagents are used to drive reactions tocompletion. Because of the speed and simplicity of the repeated steps,the major portion of the solid-phase procedure is amenable toautomation. Once chain elaboration has been accomplished, it isnecessary to release (cleave) the crude peptide from the support underconditions that are minimally destructive towards sensitive residues inthe sequence.

Antibody Binding Moieties

Other polypeptide binding moieties include immunoglobulin bindingmoieties that include at least one immunoglobulin domain, and typicallyat least two such domains. An “immunoglobulin domain” refers to a domainof a antibody molecule, e.g., a variable or constant domain. An“immunoglobulin superfamily domain” refers to a domain that has athree-dimensional structure related to an immunoglobulin domain, but isfrom a non-immunoglobulin molecule. Immunoglobulin domains andimmunoglobulin superfamily domains typically include two β-sheets formedof about seven β-strands, and a conserved disulphide bond (see, e.g.,Williams and Barclay 1988 Ann. Rev Immunol. 6:381-405). Proteins thatinclude domains of the Ig superfamily domains include T cell receptors,CD4, platelet derived growth factor receptor (PDGFR), and intercellularadhesion molecule (ICAM).

One type of immunoglobulin binding moiety is an antibody. The term“antibody,” as used herein, refers to a full-length, two-chainimmunoglobulin molecule and an antigen-binding portion and fragmentsthereof, including synthetic variants. A typical antibody includes twoheavy (H) chain variable regions (abbreviated herein as VH), and twolight (L) chain variable regions (abbreviated herein as VL). The VH andVL regions can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (CDR), interspersed withregions that are more conserved, termed “framework regions” (FR). Theextent of the framework region and CDR's has been precisely defined(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917). Each VH and VL is composed of three CDR's and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

An antibody can also include a constant region as part of a light orheavy chain. Light chains can include a kappa or lambda constant regiongene at the COOH-terminus (termed CL). Heavy chains can include, forexample, a gamma constant region (IgG1, IgG2, IgG3, IgG4; encoding about330 amino acids). A gamma constant region can include, e.g., CH1, CH2,and CH3. The term “full-length antibody” refers to a protein thatincludes one polypeptide that includes VL and CL, and a secondpolypeptide that includes VH, CH1, CH2, and CH3.

The term “antigen-binding fragment” of an antibody, as used herein,refers to one or more fragments of a full-length antibody that retainthe ability to specifically bind to a target. Examples ofantigen-binding fragments include, but are not limited to: (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsoencompassed within the term “antigen-binding fragment.”

Antibody Production and Isolation

Typically, an immunoglobulin binding moiety is monospecific.Monospecific antibodies can be obtained by cloning and expressingantibody genes, e.g., from a monoclonal antibody cDNA. Also, polyclonalantibodies can be generated by immunization of, e.g., a horse, goat,rabbit, sheep, with an antigen. Production of antibodies and antibodyfragments is well documented in the field. See, e.g., Harlow and Lane,1988. Antibodies, A Laboratory Manual. Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory. For example, Jones et al., Nature 321: 522-525(1986), which discloses replacing the CDRs of a human antibody withthose from a mouse antibody. Marx, Science 229: 455-456 (1985),discusses chimeric antibodies having mouse variable regions and humanconstant regions. Rodwell, Nature 342: 99-100 (1989), discusses lowermolecular weight recognition elements derived from antibody CDRinformation. Clackson, Br. J. Rheumatol. 3052: 36-39 (1991), discussesgenetically engineered monoclonal antibodies, including Fv fragmentderivatives, single chain antibodies, fusion proteins chimericantibodies and humanized rodent antibodies. Reichman et al., Nature 332:323-327 (1988) discloses a human antibody on which rat hypervariableregions have been grafted. Verhoeyen, et al., Science 239: 1534-1536(1988), teaches grafting of a mouse antigen binding site onto a humanantibody.

Polysaccharide Binding Moieties

In certain embodiments, the binding moiety is a polysaccharide, linked,using any of a variety of chemistries, by a single bond, e.g., acovalent bond, at one of the two ends, to a functional group on thenanoparticle. The polysaccharides can be synthetic or natural. Mono-,di-, tri- and polysaccharides can be used as the binding moiety. Theseinclude, e.g., glycosides, N-glycosylamines, O-acyl derivatives,O-methyl derivatives, osazones, sugar alcohols, sugar acids, sugarphosphates when used with appropriate attachment chemistry to thenanoparticle.

A generally useful method of accomplishing linking is to couple avidinto a magnetic nanoparticle and react the avidin-nanoparticle withcommercially available biotinylated polysaccharides, to yieldpolysaccharide-nanoparticle conjugates. For example, sialyl Lewis basedpolysaccharides are commercially available as biotinylated reagents andwill react with avidin-CLIO (see Syntesome, Gesellschaft fürmedizinische Biochemie mbH.). The sialyl Lewis x tetrasaccharide(Sle^(x)) is recognized by proteins known as selectins, which arepresent on the surfaces of leukocytes and function as part of theinflammatory cascade for the recruitment of leukocytes.

Still other targeting moieties include a non-proteinaceous element,e.g., a glycosyl modification (such as a Lewis antigen) or anothernon-proteinaceous organic molecule.

Polysaccharide Isolation

Bacterial membrane-attached polysaccharide can be purified, for example,from the SKU 1100 strain by cell disruptions using either ultrasonictreatment or lysozyme treatment, followed by ultracentrifugation, enzymetreatments, dialysis against SDS, DEAE-cellulose column chromatography,alcohol precipitation, and gel filtration chromatography.

Polysaccharides can also be synthesized and are commercially available.

Coupling of Binding Moieties to Nanoparticles to Prepare Conjugates

The conjugates are prepared by linking two or more binding moieties toeach magnetic nanoparticle. A general procedure for synthesizingamino-cross linked iron oxide nanoparticle begins with the synthesis ofa dextran coated superparamagnetic iron oxide. There are a variety ofsatisfactory procedures which can employed such as those in Weissleder“Monocrystalline iron oxide particles for studying biological tissues”U.S. Pat. No. 5,492,814; Molday, R. S. and D. MacKenzie, “Immunospecificferromagnetic iron-dextran reagents for the labeling and magneticseparation of cells,” J. Immunol. Methods, 1982, 52(3):353-67, Palmacci“Synthesis of Polysaccharide Coated Superparamagnetic Oxide Colloids,”U.S. Pat. No. 5,262,176.

For example, a pure dextran coated superparamagnetic iron oxide can bereacted with a crosslinking agent such as 5-50% epichlorohydrin orepibromohydrin in strong base (final concentration 1-3 M NaOH). After asufficient time at room temperature, liquid ammonia in excess is addedto aminate the polysaccharide. Low molecular weight impurities areremoved, e.g., by centrifugation or exhaustive ultrafiltration using amembrane with a 10 kDa cutoff.

Coupling of Oligonucleotides to Nanoparticles

The invention provides for preparing oligonucleotides with reactive 3′,5′, or both termini. One terminus is attached to the surface of thenanoparticle, leaving the other terminus free for attachment to anothermolecule, e.g., a biotin group or another tag.

Table 1 provides a partial list of techniques and reagents that can beused to couple oligonucleotides to amino- or carboxy-functionalizednanoparticles. The general strategy is to provide an oligonucleotidewith a unique reactive group on the 3′ or 5′ end. Exemplary groupsinclude sulfhydryl, amino, and phosphate groups. Oligonucleotides withsulfhydryl groups at the 3′ or 5′ end are of particular value, and arecommercially available. They can be coupled to amino-nanoparticlesthrough the use of reagents such as N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) and long chain SPDP (lc-SPDP) thatproduce a cleavable disulfide bond between the nanoparticle and theoligonucleotide. Amino-nanoparticles can also be reacted with reagentssuch as succinimidyl-iodoacetate to produce non-cleavable bonds betweenthe nanoparticle and oligonucleotide.

TABLE 1 Functional Groups and Strategies for coupling oligonucleotidesto nanoparticles Oligonucleotide Nanoparticle Coupling Terminal GroupFunctional Group Chemistry Cleavable Sulfhydryl Amino SPDP, lc-SPDP Yes(lc, long chain) Sulfhydryl Amino Succinimidyl- No iodoacetate AminoCarboxyl CDI No (carbodiimide) Phosphate Amino CDI No Biotin Avidin Notapplicable Not applicable

Thus, nanoparticles can be conjugated to oligonucleotides through avariety of conjugation chemistries. See U.S. Pat. No. 5,512,439; GregHermanson “Bioconjugate Techniques,” Academic Press, 1996; GordonBickerstaff “Immobilization of Enzymes and Cells,” Humana Press, 1997.If a colloid containing a variety of sizes results, particles can befractionated according to size, e.g., by ultrafiltration.

Non-polymeric surface functionalized metal oxides are coupled tooligonucleotides using coupling chemistries as shown, for example, inTable 1.

In other embodiments, populations of nanoparticle conjugates can besynthesized by allowing biotinylated oligonucleotides, polypeptides, orpolysaccharides, to react with avidin (or streptavidin)-boundnanoparticles. Here a non-covalent, but tight, bond between thebiotinylated binding moiety, e.g., oligonucleotide, and avidin of thenanoparticle attaches the oligonucleotide to the nanoparticle.Oligonucleotide-nanoparticle conjugate populations prepared in thisfashion are analogous to those prepared with covalent chemistries (Table1), and can be reacted with target oligonucleotides. Specific bindingligand pairs other than avidin-biotin are well known, and can also beused, e.g., fluorescein and antibodies specific for fluorescein, peptidehormones and their receptors, and steroids and their receptors, as longas they do not interfere with the function of the binding moieties.

An alternative protocol involves allowing two biotinylatedoligonucleotides to react with (e.g., hybridize to) a targetoligonucleotide. Following this reaction, a cross-linked iron oxide(CLIO) particle linked to avidin or streptavidin is added. The presenceof the target nucleotide again results in the formation of aggregatesand changes in T2. In this case, two populations ofoligonucleotide-nanoparticle conjugates are formed when theavidin-nanoparticle is reacted with two biotinylated oligonucleotides.An advantage of this indirect capture method is that the biotinylatedoligonucleotides that react with a target oligonucleotide are farsmaller, and hence react faster, than oligonucleotide-nanoparticleconjugates. Biotinylated-oligonucleotides have molecular weights lessthan 50 kDa, while oligonucleotide-nanoparticle conjugates havemolecular weights greater than about 1000 kDa (e.g., 1000, 2000, 3500,5000, or more up to about 10,000 kD).

An alternative to the avidin-biotin system is the use of two-dye labeledoligonucleotides, which hybridize to a target oligonucleotide. Anantibody to the dye coupled to a CLIO is then added.

Coupling of Polypeptides and Antibodies to Nanoparticles

The invention provides for preparing polypeptides with reactive 3′, 5′,or both termini. One end is linked to the surface of the nanoparticle,leaving the other end free for attachment to another molecule, e.g., abiotin group or another tag.

The conjugation of polypeptides to nanoparticles can be accomplished bya large number of conjugation chemistries and reagents some of which arealso used for attaching oligonucleotides to nanoparticles, see Table 1.A preferred general strategy is to use one of the large number ofbifunctional agents that can be reacted first with the amino group ofthe nanoparticle, and secondly with the thiol group of the polypeptide(or biomolecule). Examples of such bifunctional reagents are SPDP, MBS,lc-SPDP and SMCC and are available from companies, e.g., PierceChemical, Molecular Probes or Molecular Biosciences. The bifunctionalagent is dissolved in DMSO and reacted in excess with the aminofunctionalized nanoparticle at pH 8 using a non-amine containing buffer(e.g., borate, phosphate). Unreacted bifunctional agent is removed bydialysis, ultrafiltration, gel permeation chromatography or by usingmagnetic filters. The sulfhydryl bearing polypeptide (biomolecule) isthen added and allowed to react. Unreacted polypeptide can be removed bythe separation methods above. For details see Josephson et al, (1999)High-efficiency intracellular magnetic labeling with novelsuperparamagnetic-Tat peptide conjugates, Bioconjugate Chemistry, 10,186-91; Perez et al. (2002) DNA-based magnetic nanoparticle assemblyacts as a magnetic relaxation nanoswitch allowing screening ofDNA-cleaving agents, Journal of the American Chemical Society, 124,2856-2857; Kang et al. (2002) Magnetic Resonance Imaging of InducibleE-Selectin Expression in Human Endothelial Cell Culture, BioconjugateChemistry, 13, 122-127; Hoegemann et al, (2000) Improvement of MRIProbes To Allow Efficient Detection of Gene Expression, BioconjugateChemistry, 11, 941-946. Detailed protocols are also available from themanufacturers.

In one embodiment, the conjugate can be synthesized by allowing abiotinylated antibody or antibody fragment to react with avidin (orstreptavidin) nanoparticles. Here a non-covalent, but tight, bondbetween the biotinylated antibody and avidin of the nanoparticleattaches the antibody to the nanoparticle.

In another embodiment, a natural or synthetic polypeptide is covalentlyor non-covalently attached to the nanoparticle while the other terminalis biotinylated.

In one aspect of the invention, both ends of the polypeptide arebiotinylated and avidin is directly attached to the nanoparticle.

In another embodiment, both termini of the peptide are covalently ornon-covalently attached to two nanoparticles.

Coupling of Polysaccharides to Nanoparticles

The invention provides for preparing polysaccharides with reactive ends.One end is attached to the surface of the nanoparticle, leaving theother end free for attachment to another molecule. For example, asdescribed above, the free end of the polysaccharide can be biotinylatedand aggregation can be induced by exposure to avidin. Also, thepolysaccharide can be biotinylated on both termini and exposed to avidinlinked to a nanoparticle.

Characterizing Conjugates

The conjugates can form several conformations, or “states,” in solution.The first is the monodispersed conformation, represented when a bindingmoiety of a conjugate has not reacted with a target molecule. Thisconformation is approximately 4-100 nm (e.g., 5, 10, 25, 40, 50, 75, or90 nm) in size.

The second conformation is a small aggregate, which contains 2 to about20 (e.g., 3, 5, 7, 10, 15, or 20) individual nanoparticle conjugatesheld together by the interaction (e.g., binding) of the binding moietywith a target, or with another binding moiety. The association of thenanoparticles is mediated by the attached biomolecules and not bynanoparticle non-specific attractions. This aggregate is approximately100-500 nm (e.g., 200, 250, 300, or 400 nm) in size, is stable, andremains in solution. The metal oxide, e.g., iron oxide, concentrationused to form the small aggregate is about 1-25, e.g., 5-20 μg/ml. Thesmall aggregates do not settle out of solution and are “porous” in thatthey do not sterically block large molecules (e.g., enzymes) fromentering the aggregate. The “pores” are really spaces or openingsbetween the binding moieties that combine to form the aggregates, whichcan be envisioned as a three-dimensional lattice or mesh. The size ofthe openings can be controlled by adjusting the size of thenanoparticles and the size of the binding moieties on each conjugate.The small aggregates are stable under a variety of conditions, e.g.,stable from 4° C. to 80° C., stable in denaturants, stable in highsalts, and is stable at a pH varying from about 5.5 to 14.

The third conformation is the large aggregate cluster, which is, ineffect, an aggregate of aggregates. The cluster contains greater than 20nanoparticles and is greater than 500 nm in size. The cluster is notuseful since it typically clumps and falls out of solution.

The nanoparticle conjugates can be used as magnetic nanosensors ormagnetic relaxation switches (MRS) in various detection systems. Forexample, the new methods can utilize detectors that measure the magneticproperties of the conjugates and aggregates (e.g., magnetometers,oscillating magnetic field readers, and superconducting quantuminterference device (SQUID) detectors). Other detection methods includemagnetic force microscopy or atomic force microscopy, flow cytometry,centrifugation, light scattering, and size separation.

In magnetic resonance (MR) imaging applications, the novel conjugatesprovide methods for the detection and a spatial localization of specificsequences of target molecules, such as nucleic acids, in living systems.This is based on the remarkable ability of the magnetic conjugates toeffect water relaxation in a sequence specific manner even in media thatwill not permit assays using light-based methods of gene detection (seeExample 5). Hence, the new conjugates can function as MR contrast agentsfor the detection of target molecules, such as nucleic acids andpolypeptides in vivo.

The new conjugates are essentially nontoxic to mammalian cells. In oneembodiment, non-degradable oligonucleotide analogs (e.g., peptidenucleic acid or PNA) may be coupled to nanoparticles and used to imagesequences of nucleic acids in vivo. Nontoxicity is evident from the useof magnetic nanoparticles as the active ingredient of COMBIDEX®, ananoparticle-based MR contrast agent, which has been judged approvableby the FDA (January 1999). COMBIDEX® is similar to MION (monodisperseiron oxide nanospheres). MION is a starting material for aminated CLIOused in one embodiment herein (see FIG. 1.). Thus, the new conjugatesand aggregates can be administered to a subject, e.g., a human oranimal, such as a mammal (e.g., dogs, cats, cows, pigs, and horses).Various routes of administration can be used to achieve systemic orlocal delivery. Because of the specific binding characteristics

Oligonucleotide Conjugates

The starting material used in some of the examples described hereinconsists of monodisperse or polydisperse, fluid-phase nanoparticlescontaining superparamagnetic Fe₂O₃/Fe₃O₄ (3-5 nm), caged byepichlorohydrin cross-linked dextran, and functionalized with aminegroups (NH₂-CLIO). Thiolated oligonucleotides were coupled to NH₂-CLIOusing N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as a linker(FIG. 1). The physical properties of the conjugates P1(CLIO-SS-((CH₂)₆-CGC-ATT-CAG-GAT) (SEQ ID NO:1)) and P2((TCT-CAA-CTC-GTA-(SEQ ID NO:2)(CH₂)₃)-SS-CLIO) are summarized in Table2. P1 and P2 each had an average of 3 oligonucleotides per particlebased on a single crystal per particle and 2064 iron atoms per crystal(see Shen reference above). They could be stored at room temperature or4° C. for several months without precipitation.

To effect maximum detection of target oligonucleotide sequence,hybridization conditions are established by methods well known in theart. Hybridization of the oligonucleotide-nanoparticle conjugates to thetarget nucleic acids is typically performed under moderate to highstringency conditions. The parameters of salt concentration andtemperature, which affect stringency of hybridization, can be varied toachieve the optimal level of identity between the base sequences of theoligonucleotide-nanoparticle conjugates and those of the targetoligonucleotide or nucleic acid being detected. These techniques andmethods are well-known in the field. Additional guidance regarding suchconditions is available, for example, in Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.;and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology,(John Wiley & Sons, N.Y.) at Unit 2.10.

For example, if stringent hybridization conditions are desired, one canperform hybridization in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50°C. Another example of stringent hybridization conditions ishybridization in 6×SSC at about 45° C., followed by one or more washesin 0.2×SSC, 0.1% SDS at 55° C. A further example of stringenthybridization conditions is hybridization in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. Otherstringency conditions are 0.5M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

To demonstrate the ability of P1 and P2 to hybridize to a targetoligonucleotide, P1/P2 mixtures were incubated with a complementaryoligonucleotide. The samples became slightly turbid within 3-4 hours atroom temperature, with a brown precipitate forming after 16 hours (FIG.2B). The precipitate in the vial shown in FIG. 2B was moved to the sidewith a hand held magnet. Tubes containing the P1/P2 mixture alone, theP1 with complementary oligonucleotide, or the P1/P2 mixture withhalf-complementary oligonucleotide did not show turbidity or precipitateformation even after weeks at room temperature (FIGS. 2A, 2C, and 2D).

To further elucidate the interaction of P1/P2 with complementaryoligonucleotide, gel electrophoresis was performed. Under non-denaturingconditions, and without DTT, the precipitate (as shown in the vial inFIG. 2B) remained at the top of the gel (FIG. 3A, lane 1). Treatmentwith DTT (FIG. 3A, lane 2) resulted in a single band of hybridizedoligonucleotide. Under denaturing conditions and with DTT added (FIG.3B), two bands were observed, the slower one consisting of complementaryoligonucleotide and the faster one of a mixture of 3′ and 5′oligonucleotides.

The nanoparticles P1 and P2 are potent enhancers of the spin-spin andspin-lattice relaxation processes (Table 2). Interestingly, thespin-spin relaxation was furthermore significantly enhanced byoligonucleotide hybridization, rendering the particles as potential“magnetic nanosensors.” FIG. 4 shows the effect of oligonucleotideaddition to an aqueous solution of P1/P2. Within several minutes, T2decreased from 63 ms to 45 ms and this effect persisted for the periodof observation (2 hours). The insert in FIG. 4 plots the T2 decrease asa function of oligonucleotide added. Table 2 summarizes theconcentration independent R1 and R2 relaxivities before and afterhybridization. Hybridization primarily affected R2 with a doubling ofthe R2/R1 ratio. Concomitant laser light scattering indicated asignificant size increase of hybridized conjugates, presumably causingeffects on spin-spin relaxation.

TABLE 2 Size and relaxivities before and after hybridization with targetoligonucleotides R1 ** R2 ** Hybrid- Size (sec⁻¹ (sec⁻¹ Compound ization(nm) mM⁻¹) mM⁻¹) R2/R1 P1 No 53 ± 11 27.7 ± 0.3 75 ± 2 2.7 ± 0.1 P2 No53 ± 11 26.7 ± 0.3  71 ± 10 2.6 ± 0.4 P1 + P2 + No 65 ± 25 25.8 ± 0.4 67± 1 2.6 ± 0.1 oligo non- complement. P1 + P2 + Yes 215 ± 19  23.0 ± 1.0128 ± 3  5.6 ± 0.2 oligo com- plement. * Determined by gelelectrophoresis ** Relaxivities (units of sec⁻¹ mM⁻¹) are the slopes ofplots of relaxation rate (1/T, sec⁻¹) against nanosensor concentration(Fe, mM); values are plotted as means ± SD, n = 3. Size refers to theunimodal size distribution as determined by light scattering, mean ± SD,n = 6.

The effect of temperature cycling on the hybridization of theoligonucleotide nanoparticle was investigated by measuring changes in T2values (FIG. 5). At 80° C., hybridization was minimal and T2 changeswere small. During multiple cycles of heating and cooling representativeT2 changes were observed. Furthermore, upon addition of DTT,oligonucleotides were cleaved from the nanoparticles and T2 did notchange during further temperature cycling. These results indicate thatoligonucleotide hybridization efficiently changes the spin-spinrelaxation time of water, that these effects occur within minutes, thatthe magnetic effects are fully reversible through the use of DTT.

Selectivity

A unique feature of the magnetic nanoparticles is that they are highlystable to temperature fluctuations and to different ionic media. Thisstability enabled the use of buffer conditions (e.g. 25 mM KCl, 50 mMTris, pH 7.5) in which small differences in base pairing can be detectedby T2 measurements (Table 3). Thus, the new conjugates are highlyselective in binding to target molecules. For example, a singlenucleotide insertion in the center of a target sequence can abrogatemagnetic switching nearly completely. Similar effects are also seen withlarger and other kinds of single and double inserts (Table 3).

To determine the effect that nucleotide mismatches could have on T2measurements, additional target sequences containing both single andmultiple mismatches were tested. Again, single nucleotide mismatcheswere detectable while double mismatches completely abrogated magneticswitching (Table 3).

TABLE 3  Summary of tested oligonucleotide sequencesand magnetic measurements Deviation ∂T2 from p- (msec)* normal ** valuePerfect match TAC-GAG-TTG-AGA-ATC-CTG- 30 ± 2  NA NAAAT-GCG SEQ ID NO: 3 (match) Insertions AC-GAG-TTG-AGA-G-ATC-CTG- 5 ± 183% 0.0001 AAT-GCG SEQ ID NO: 4 TAC-GAG-TTG-AGA-GAG-TGC-   2 ± 0.6 93%0.0001 ATC-CTG-AAT-GCG SEQ ID  NO: 5 TAC-GAG-G-TTG-AGA-ATC-CTG-   4 ±2.5 87% 0.0002 AAT-GCG SEQ ID NO: 6 TAC-GAG-G-TTG-AGA-ATC-CTG-   2 ± 0.593% 0.0001 G-AAT-GCG SEQ ID NO: 7 Mismatches TAC-GAG-TTG-AGA-CTC-CTG- 21 ± 1.2 30% 0.0029 AAT-GCG SEQ ID NO: 8 GAC-GAG-TTG-AGA-ATC-CTG-  21 ±0.6 30% 0.0020 AAT-GCG SEQ ID NO: 9 TAC-GAG-TTG-AGA-ATC-CTG-  15 ± 0.650% 0.0030 CAT-GCG SEQ ID NO: 10 TAC-GAG-TTG-AGA-CTC-CTC-   1 ± 0.6 97%0.0001 AAT-GCG SEQ ID NO: 11 TAC-GAC-TTG-AGA-ATC-CTG-   9 ± 1.7 70%0.0002 CAT-GCG SEQ ID NO: 12 *∂T2 = T2_((t−0 min)) − T2_((t−30 min)); **deviation =(∂T2_((perfect match)) −∂T2_((insertion or mismatch))/∂T2_((perfect match))) × 100

The selectivity of the MRS was further studied by preparing probes totarget a GFP gene sequence and three variants with a single mismatch (T,C, G instead of an A). FIG. 8A shows the time course of T2 measurementswith these four sequences. The perfect match (containing A) decreased T2within minutes of oligonucleotide addition. The single mismatchesbehaved differently and these differences could be readily detected byT2 measurements at 40° C. As in the previous experiments, we alsoperformed MR imaging at room temperature and were able to show similardifferences (FIG. 8B). These data show that selective measurementscapable of distinguishing single nucleotide mismatches can be carriedout reliably and at various temperatures using either NMR or MMtechniques without the need for melting curve analysis.

Polypeptide Conjugates

Polypeptide conjugates behave in the same manner as the oligonucleotideconjugates, in that they are highly selective in their binding to targetmolecules and form aggregates as described further herein.

Uses of Binding Moiety-Nanoparticle Conjugates

The new conjugates can be used in two broad applications. In oneapplication, the aggregate formation assay, a population of conjugates(or a mixture of two or more populations of conjugates with differingbinding moieties directed to the same target molecule or type of targetmolecule) is placed into a sample solution. In this assay system, if thesample solution contains a target molecule to which the binding moietiesspecifically bind, the binding moieties interact with and bind to thetarget molecule resulting in the formation (self-assembly) ofaggregates. As a result, the dispersed state of the conjugates switchesto an aggregated state, which decreases T2 relaxation times. FIG. 1depicts one embodiment of such an interaction in which two conjugates,P1 and P2, combine to form an aggregate of six conjugates.

In the other application, the aggregate dispersion assay, conjugates areused to prepare small aggregates, and the aggregates are placed into asample solution. In this assay system, the binding moieties are designedso that they can be bound to each other (or to a specific aggregationinducing molecule, such as avidin) to form the aggregates, and to be (orform upon binding to each other or to the aggregation inducing molecule)a substrate that is cleaved by a specific target molecule. If the samplesolution contains a target molecule, the substrate formed by the bindingmoieties is cleaved, resulting in the dissolution of the aggregates.Thus, the aggregated state switches to a dispersed state, whichincreases T2 relaxation times.

These aggregates can be observed and detected in vitro, e.g., in vialsor arrays, e.g., 2-D or 3-D arrays, as well as in vivo, e.g., using MRimaging of a subject after administration of the conjugates oraggregates.

Aggregate Formation Assays

In this application, the conjugates must include binding moieties thatspecifically bind to at least two different binding sites or epitopes onthe target molecule, and each conjugate must have at least two bindingmoieties. In general, the conjugates are added to a sample solutionunder conditions that enable the binding moieties to interact with andbind to the target molecule. As more of these interactions occur overtime, several conjugates will accumulate together to form one aggregate.The endpoint of the assay is the detection of the presence of theaggregates, e.g., using MR imaging or other detection methods.

This application of the invention can be used in several assay systems.For example, when the binding moieties are nucleic acids, the newconjugates can be used for an analytic method referred to herein as theHybridization Relaxation Assay System or HYRAS. Like the earlier SMRAS(Solvent Mediated Relaxation Assay System) technology (U.S. Pat. No.5,164,297), HYRAS can be used to determine the concentration of ananalyte in a sample by monitoring changes in a solvent relaxation rate.HYRAS differs from SMRAS in a number of important ways.

First, HYRAS involves the assay of nucleic acids using superparamagneticiron oxide nanoparticles, and is based on the observation that nucleicacids do not non-specifically adsorb to iron oxides. This is surprisingbecause the affinity between the iron on the surface of the iron oxideand phosphate or phosphate-containing compounds, such as nucleic acids,is strong. For example, iron oxides have been used to bind and extractDNA (see, e.g., examples 10 and 11 in U.S. Pat. No. 5,512,332). Thus, itis surprising that oligonucleotides, which contain a multiplicity ofphosphate groups, do not interact non-specifically with the iron oxidenanoparticles.

Second, to produce the needed aggregation of nanoparticles by a specifictarget nucleotide, two types of oligonucleotide-nanoparticles areneeded, each with a single type of oligonucleotide attached, eachreacting with a different sequence present on a target complementaryoligonucleotide (see FIG. 1). If two different oligonucleotides werecoupled to the sample nanoparticle, the target nucleic acid wouldhybridize to the oligonucleotides on the same particle and no effect onwater relaxation rates (T2) would result. SMRAS has no requirement forthe synthesis of two different types of magnetic particles. InsteadSMRAS uses multivalent proteins coupled to an iron oxide, which thenreacts with multivalent proteins such as antibodies to produce changesin relaxation.

Third, in SMRAS and HYRAS particle aggregation alters T2 in oppositemanners. For example, in SMRAS, the complex between BSA-coated magneticparticles and anti-BSA antibodies causes an increase in T2 (decrease in1/T2); addition of BSA blocks this effect increasing 1/T2, see FIG. 4 ofU.S. Pat. No. 5,164,297. In contrast, in HYRAS, whenoligonucleotide-nanoparticles react with a target nucleotide to formaggregates there is a decrease in T2.

Fourth, the conjugation strategy used in HYRAS differs from that used inSMRAS. In HYRAS, oligonucleotides are attached to iron oxide colloidpolymers with a single covalent bond at the 3′ or 5′ end of theoligonucleotide. This is essential because the oligonucleotides aresufficiently small (short), that if they were attached in the middle,and not by their 3′ or 5′ ends, they would not be able to hybridize totarget nucleotides. In contrast, particles used in SMRAS, such as thedextran-coated iron oxide or BSA-coated iron oxide, are synthesized bythe adsorption of polymers to the surface of the iron oxide. Thisattachment is maintained by a large number of non-covalent bonds.

The new methods can also be used to detect nucleic acids by magneticresonance. This assay measures the presence of target oligonucleotidesin turbid or tissue-like samples (see Example 5). This method providesadvantages over light-based analytical methods, such as thenon-magnetic, gold based colorimetric assays described in WO 98/04740.In one method using the gold nanoparticles, the color change isdetermined in solution, which requires a non-turbid, non-opaquesolution. In a second method, oligonucleotide-gold conjugates arecollected on an oligonucleotide bearing substrate, such as membrane orfilter. Excess media, which interferes with the detection, is removedand amplification by a silver stain is employed. In the presentinvention, neither separation nor amplification steps are used. Instead,the presence of nanoparticle aggregate is detected by MR. The inventioncan be distinguished by the ability to “see” aggregate formation inhighly turbid or opaque tissues by the use of magnetic resonance. Thisyields assays with reduced processing and handling steps.

The novel conjugates of the invention can be used to measure the T2values, and levels of oligonucleotides, in several samplessimultaneously. This can be accomplished by replacing an MR spectrometer(FIGS. 4, 5 and 6 with an MR imager (FIG. 7). MR signal intensity wasdetermined with a T2 weighted pulse sequence for a matrix of 24 wells ofa 384 well microtiter plate. Further reduction in sample size, forexample by the use of 1534 well microtiter plates, can be achieved.Microtiter plates can be stacked, and the capability of MR to measurethe signal intensity of many slices, i.e., in three-dimensions, can beused to further increase assay throughput.

The new conjugates can also be used as MR contrast agents. In oneembodiment, dextran coated superparamagnetic iron oxides (MION orCOMBIDEX®) are synthesized (see U.S. Pat. Nos. 5,492,814 or 5,262,176)and then cross-linked and amino functionalized to yield NH₂-CLIO, asdescribed herein. Alternatively, non-polymer coated iron oxide particlescan be used. The nanoparticles are then coupled to specificoligonucleotides as shown, e.g., in FIG. 1. The resultingoligonucleotide-nanoparticle conjugates are then formulated in aphysiologically acceptable media (e.g., saline or isotonic mannitol) andinjected into an animal or human, intravenously at a dose between 0.1and 10 mg Fe/kg. The contrast agent is permitted to accumulate in targettissue and is detected at highest sensitivity with T2 weighted spin-echoor gradient-echo pulse sequences.

In another example, detection of an mRNA in solution can be accomplishedby synthesizing two populations of conjugates. The first contains anoligonucleotide sequence complementary to a sequence in the mRNA ofinterest and is bound at the 3′ or 5′ termini to the nanoparticle. Asecond conjugate is synthesized with a oligonucleotide sequencecomplementary to a different but proximate sequence of the mRNA.Addition of these conjugates to a solution containing the mRNA willresult in the binding of the conjugates resulting in aggregation of theconjugates. Aggregation will produce a measurable decrease in the T2 byMR technology.

These novel conjugates can be used to determine the pattern of geneexpression in a specimen (expression analysis) by extension of themethods shown in Example 6, below. Here a microtiter plate is preparedwhere each well contains different combinations ofoligonucleotide-nanoparticles, i.e., combinations of oligonucleotideswith different sequences attached to the same magnetic nanoparticle. Thesequences of the oligonucleotides are chosen to permit hybridization,followed by aggregation and T2 change, with a unique target sequencethat may or may not be present in the sample.

Another embodiment uses the same concept, but with proteins. Forexample, the conjugate can be used to detect the presence of an antigenin a sample. In this method, antibodies are linked covalently ornon-covalently to the nanoparticle. To ensure that the antigen bindingsite is exposed, the C-terminus of the antibody or antibody fragment isattached to the nanoparticle. Monoclonal antibodies can be used for thismethod. A feature of this method is the need for a mixture of at leasttwo types of nanoparticles, each with a specific binding moiety, e.g.,monoclonal antibody attached. The antibodies are directed toward thesame antigen, but recognize different determinants or epitopes. Thepopulations are mixed in a sample and binding of the conjugate to anantigen induces aggregation, resulting in a measurable decrease in T2.

In another aspect of the invention, a polyclonal antibody can beattached to the nanoparticle. Since by definition these antibodies aremultivalent, only a single population of conjugates is required.

Antibody fragments can also be used as long as they are bivalent. Ifsingle chain FIT fragments are used, there must be two populations ofconjugates prepared. Each population will contain a single chainfragment directed to a distinct epitope of the same antigen.

These conjugates can also be used, as described above for theoligonucleotides, as magnetic nanosensors in other methods of antigendetection systems. These methods can utilize detectors that measure themagnetic properties of the particles (e.g., magnetometers, oscillatingmagnetic field readers, and superconducting quantum interference device(SQUID) detectors). Other detection methods include magnetic forcemicroscopy or atomic force microscopy.

In the MR imaging application, the novel conjugates provide a method forthe detection and a spatial localization of specific antigens in livingsystems. Hence antibody conjugates can function as MR contrast agentsfor the detection of polypeptides in vivo.

In another embodiment, conjugates can be useful in detecting a targetmolecule, e.g., an antibody, in solution. In this assay, the antigenwill be bound to the nanoparticle and placed into a sample. If anantibody directed to the antigen is present, binding of the antigen willcause aggregation of the conjugate resulting on the decrease of T2. Thisassay method can be used for polyclonal and monoclonal antibodies andantibodies of any subclass because of the bivalent or polyvalent natureof the antibodies. This assay method can be used in the detection ofantibodies, for example, in serum, acites fluid, cell culture medium,and cell lysates.

In another embodiment, the binding moiety can be a receptor-bindingprotein bound to the nanoparticle. When applied to a solution of cells,clustering of a cell surface receptor will result in aggregation of theconjugate followed by the concomitant decrease in T2. In another aspectof the invention, a kinase activity can be assayed. A peptide sequencewith a serine or tyrosine kinase recognition site is attached to ananoparticle at one terminal end. Addition of a solution containing akinase will result in the phosphorylation of the binding moiety.Exposing the conjugates to anti-phosphotyrosine or anti-phosphoserineantibody will result in aggregation resulting the decrease of T2

Aggregate Dispersion Assays

In this application, a change in T2 is measured by preparing anaggregate of several conjugates, and then placing the aggregate into asolution (resulting in an immediate decrease in the T2), which maycontain a target molecule. The aggregate is prepared by designing thebinding moieties to form a substrate that is cleaved by the targetmolecule, thus dispersing the aggregates into conjugates, resulting inan increase of the T2 relaxation time. The binding moieties can bind toeach other to form the substrate, or can contain the substrate, and formthe aggregates by binding to an aggregation inducing molecule, such asavidin. The endpoint of the assay is the detection of the dissolution ordispersal of the aggregates (or the lack of formation of an aggregate ifthe target molecule and aggregate forming molecule are added to asolution of the conjugates at the same time).

In one embodiment, the new methods can be used to detect enzyme targetmolecules in a sample solution. The assay is based on the attachment tothe nanoparticle of a natural or synthetic peptide that has an internalenzymatic site. Biotin is attached to the free terminus of the peptide.These biotin-labeled conjugates are mixed into a solution, and avidin(which binds four molecules of biotin per molecule of avidin) is addedas the aggregation inducing molecule to form aggregates. Theseaggregates are then added to a sample solution. If there is a measurableincrease in T2, then the enzyme is present. Alternatively, theconjugates can be added to a sample solution along with the avidin. Ifbinding results in the aggregation of the conjugates, a measurabledecrease in T2 can be observed, indicating that the target proteolyticenzyme is not present. However, if the enzyme is present and reacts withthe substrate peptide in a relatively slow manner, a decrease in the T2can be observed, followed by an increase of T2 when the aggregates aredispersed. If the reaction is fast, no decrease in T2 will be observed.This method can be used for any hydrolase that has a known recognitionsequence.

In another variation of the invention, a peptide binding moietycontaining an internal hydrolytic sequence can have biotin attached toboth termini. Avidin is attached to the nanoparticles and mixed with thebiotinylated peptide in a sample. Since one avidin molecule binds fourbiotin molecules, aggregation will occur if the biotinylated molecule isintact or if it has been cleaved. However, the degree of aggregationwill be greater if the molecule is intact, therefore the sample willexhibit a greater degree of decreased T2.

In another aspect of the invention, immediate aggregation is induced byattaching a nanoparticle to both termini of the peptide. The conjugateis placed in the sample and the relaxivity is measured. If the enzyme ofinterest is present, then an increase in T2 will be measured when thepeptide is cleaved.

The assay for the presence of a particular polysaccharidase can beaccomplished in the same manner as described above for the enzyme assaysusing a polysaccharide as the binding moiety.

In another aspect of the invention, the conjugate can be used to detecta molecule that is transferred to the binding moiety. For example, DNAmethyltransferase activity can be assayed. Hybridizing oligonucleotideconjugates can form a dam methylation site (GATC). The hybridizationresults in aggregation of the attached nanoparticle and a measurabledecrease in T2. Upon contact with a methylase, the adenine and cytosineare methylated. Treatment with DpnI, a restriction endonuclease thatspecifically cleaves the methylated sequence GATC, results in dispersionof the aggregates followed by a measurable increase in T2.

In one aspect of the invention, oligonucleotide conjugates aresynthesized to be complementary to each other. Upon hybridization ofthese conjugates in solution, the binding moieties form adouble-stranded nucleic acid with a unique endonuclease restriction site(e.g., EcoRI, BamHI, PvuII). Hybridization of the oligonucleotides alsoaggregates the nanoparticles attached to the oligonucleotides resultingin a decreased T2. In this case, the presence of a target endonucleasein a sample can be measured by an increase in T2 when the restrictionsite is cleaved, resulting in the dispersion of the aggregates.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Synthesis of Superparamagnetic Iron Oxide Nanoparticles

Biocompatible, fluid phase magnetic nanoparticles (NH₂-CLIO) weresynthesized as described and reacted with N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) to yield 2Py-SS-CLIO (FIG. 1). SeeJosephson et al, (1999) Bioconjugate Chemistry, 10, 186-91 and Perez etal., (2002) Journal of the American Chemical Society, 124, 2856-2857.

The first step in the synthesis of amino-CLIO is the synthesis of adextran coated superparamagnetic iron oxide. A pure dextran coatedsuperparamagnetic iron oxide is reacted with a crosslinking agent (5-50%epichlorohydrin) in strong base (final concentration 1-3 M NaOH). After24 hours at room temperature, liquid ammonia in excess is then added toaminate the polysaccharide. Low molecular weight impurities were removedby exhaustive ultrafiltration using a membrane with a 10 kDa cutoff.

Example 2: Synthesis of Oligonucleotides andAlkanethiol-Oligonucleotides

The 5′-alkanethiol-oligonucleotide (HS-(CH₂)₆-CGC-ATT-CAG-GAT (SEQ IDNO:1)) and 3′-alkanethiol-oligonucleotide (TCT-CAA-CTC-GTA(SEQ IDNO:2)-(CH₂)₃-SH) were synthesized at a 1 μmol-scale using standardphosphoramidite chemistry. The sulfhydryl groups were protected with amercaptoalkyl linker. Immediately before reaction with 2Py-SS-CLIO,oligonucleotides were deprotected with dithiothreitol (DTT) (J. J.Storhoff, R. Elghanian, R. C. Mucic, C. A. Mirkin, R. L. Letsinger, J AmChem Soc 1998, 120, 1959).

Complementary ( ^(5′)TAC-GAG-TTG-AGA-ATC-CTG-AAT-GCG^(3′))(SEQ IDNO:13), half-complementary (^(5′)TAC-GAG-TTG-AGA-GAG-TGC-CCA-CAT^(3′)⋅)(SEQ ID NO:14), andnon-complementary (^(5′)ATG-CTA-AAT-GAC-GAC-TGC-CCA-CAT^(3′))(SEQ IDNO:15) oligonucleotides were synthesized using standard phosphoramiditechemistry (underlined bases will hybridize).

Example 3: Conjugation of Nanoparticles to Alkanethiol Oligonucleotide

To 1.1 mL of 2Py-SS-CLIO (3 mg of Fe in 0.1 M phosphate buffer, pH 8.0,see example 1), 550 μg of either 5′- or 3′-alkanethiooligonucleotide wasadded and incubated overnight at room temperature. The mixture waspurified using an LS+ high gradient magnetic separation column (MiltenyiBiotec, Auburn, Calif.) equilibrated with 0.1 M phosphate buffer, pH7.5. The number of oligonucleotides attached per particle was determinedby treatment with DTT, followed by separation of iron andoligonucleotide using a microconcentrator as above. Oligonucleotideconcentration was then determined from absorbance at 260 nm, using anextinction coefficient of 1.2×10⁵ M⁻¹ cm⁻¹. The probes are denoted P1(CLIO-SS-(CH₂)₆-CGC-ATT-CAG-GAT(SEQ ID NO:1)) and P2(TCT-CAA-CTC-GTA(SEQ ID NO:2)-(CH₂)₃-SS-CLIO. To 1.1 mL of 2Py-SS-CLIO(3 mg of Fe in 0.1 M phosphate buffer, pH 8.0), 550 μg of either the5-alkanethiol-oligonucleotide (HS-(CH₂)₆-CGC-ATT-CAG-GAT(SEQ ID NO:1))or the 3-alkanethiol-oligonucleotide (TCT-CAA-CTC-GTA-(SEQ IDNO:2)(CH₂)₃-SH) were added and incubated overnight at room temperature.The next day the mixture was applied to a magnetic separations column(Miltenyi Biotec, Auburn, Calif.) equilibrated with 0.1 M phosphatebuffer, pH 7.5. The column was washed with phosphate buffer to removeany non-bound oligonucleotide.

Example 4: Assay for Target Oligonucleotide

Hybridization: To generate FIGS. 2A to 2D, equal volumes (25 μL) of P1and P2 (both at 550 μg Fe/mL) were mixed with 14 μL of 1 M NaCl, 0.1 Mphosphate, pH 7.5. Two μL (400 ng) of various oligonucleotides were thenadded. The mixture was heated to 50° C. for 5 minutes and allowed toreact at room temperature overnight. The precipitate shown in FIGS. 3Aand 3B were obtained after overnight incubation of P1/P2 withcomplementary oligonucleotide; the precipitate was washed with 0.1 MNaCl, 0.1 M phosphate buffer and re-suspended in 300 μL of the samebuffer. The sample was split into two 15 μL portions and electrophoresedwithout DTT (lane 1) or with 4 mM DTT (lane 2) under non-denaturingconditions (FIG. 3A) or denaturing conditions (FIG. 3B).

Gel electrophoresis: Non-denaturing gels (10% polyacrylamide) anddenaturing gels (20% polyacrylamide) were used after optimization of theseparation process. Gels were stained with SYBR Gold dye (MolecularProbes, Eugene Oreg.).

Determination of Proton Relaxation Times: Relaxation time measurementswere performed at 0.47 Tesla, at 40° C. (Bruker NMR Minispec, Billerica,Mass.), except for the experiment used to generate FIG. 5, wheretemperatures of 40° C. and 80° C. were used. To determine the effect ofhybridization on water T2, equal amounts of P1 and P2 (5 μL) werediluted in 1 mL 1 M NaCl, 0.1 M phosphate buffer, pH 7.5 to give a totaliron of 10 μg/mL. T2 values were obtained before and after addition of 1μL (390 ng) of complementary, half-complementary or non-complementarytarget nucleic acids and plotted as a function of time. Relaxivity wasdetermined by plotting water 1/T2 and 1/T1 as a function of ironconcentration, see Table 2. The size of conjugates was determined bylight scattering (Coulter N4, Hialeah, Fla.).

Example 5: Use of Nanoparticle Conjugates in Turbid Media

Equimolar amounts in iron of oligonucleotide-nanoparticle conjugatesdenoted P1 and P2 were diluted in a 10% Fat Emulsion (Intralipid® 10%,Baxter Healthcare Corporation) containing 1 M NaCl. Changes in water T2relaxation time were recorded after addition of 1 μL (53 femtomoles) ofcomplementary oligonucleotide. As was the case in non-turbid media (FIG.4), a rapid decrease in the T2 relaxation time is observed (FIG. 4).Three hours after adding the complementary oligonucleotide, at thearrow, 2 μL of DTT (0.4 M) was added to the P1 and P2 solution. Agradual increase in the T2 relaxation time is observed reaching theoriginal T2 value within two hours. These results indicate that the newmethods work even in turbid media.

Example 6: Array Based Assay

Equimolar amounts in iron of oligonucleotide-nanoparticles denoted P1and P2 were diluted with 1 M NaCl in 0.1 M sodium phosphate, pH 7.4 togive iron concentrations of 6 μg/mL or 3 μg/mL. 100 μL of the P1/P2mixture and 1 μL of complementary or non-complementary targetoligonucleotides were added to 24 of the square wells of a 384-wellmicrotiter plate. Images were made on a clinical MR imager (GE Signal,1.5 Tesla) using a T2-weighted pulse sequence (TR=3000 ms/TE=300 ms).FIG. 7 shows 24 wells of the 384-well plate. The top two rows contain 3μg Fe/ml and the bottom two rows contain 6 μg Fe/ml. Each column has theindicated amounts of target nucleic acids added. The wells get “darker,”i.e., the signal intensity drops because T2 drops. This is due to ahybridization-induced formation of aggregates betweenoligonucleotide-nanoparticle. No binding occurs with non-complementarytargets, and thus, there is no change in T2 and no change in signalintensity. These results illustrate the utility of the new methods foruse in in vitro arrays, which can be two- or three-dimensional.

Example 7: Assay for Green Fluorescent Protein (GFP) mRNA by Imaging MRS

To test whether MRS could be used to identify a target sequence in ahigher throughput format, a panel of cell lines was screened for GFPmRNA expression using an MR imager as a detector. The panel consisted ofprimary human and rodent tumor cells lines, one of which was transducedwith a GFP encoding HSV amplicon (Gli-36), while another one wastransiently transfected with a GFP encoding plasmid DNA (COS-1). Inaddition, the corresponding parental cell lines were transfected withbeta galactosidase as a negative control and included in the panel.Total RNA from these cell lines was isolated and imaged after sensingwith GFP-P1/GFP-P2 (FIG. 9a ). The sample in well C3 contained RNA fromthe Gli-36 cells line, whereas well D4 contained RNA from COS-1 cells.The observed magnetic changes correlated well with fluorescencemeasurements of the cell lines and RT-PCR (data not shown). Bothparental and beta galactosidase expressing Gli-36 and COS-1 cell linesdid not show a significant difference compared to wild type cell lines.

While the above experiments were carried out with isolated RNA, similarmeasurements were made in cell lysates. For these experiments, a mildlysis buffer (20 mM Tris pH 8, 5 mM MgCl₂, 0.5% NP-40, 200 μg/mL tRNA)was added to adherent cells prior to probing with P1/P2. This buffer hasbeen previously used to extract RNA from cell without the need ofscrapping the cells off the dish. As shown in FIG. 9b , differences inGFP mRNA expression between the parental and GFP expressing Gli-36 celllines were clearly identified. In additional studies, this differencewas quantified using Gli-36 cell lines infected with different MOI of aGFP bearing amplicon vector. FIG. 9c shows the correlation between cellfluorescence measurements and mRNA measurements using MRS technology incell lysates. In these studies, a specific GFP mRNA was detected in apool of total RNA (1 μg) and in whole cell lysate with no prioramplification of the signal. This level of detection with the MRStechnology is comparable to traditional fluorescent-based methods foroligonucleotide hybridization carried out with purified total RNA.

Example 8: Assay for Caspase Activity Using a Monobiotinylated Peptideand Avidin to Induce Formation of Small Aggregates

In this assay, a biotinylated peptide substrate for caspase-3 wassynthesized (Biotin-GDEVDGC caspase-3 recognition site is underlined)and coupled to aminated CLIO using SPDP (see example 1). Equimolaramounts of Avidin-CLIO and Biotinylated peptide-CLIO conjugate wereincubated in PBS (10 μg Fe/ml) to allow a small aggregate to form. TheT2 was measured before and after addition of 25 ng Caspase 3 (1.7 nM) inthe presence or absent of a caspase 3 inhibitor. See figure afterexample 10b.

The avidin-CLIO construct was made as follows: Amino-CLIO (0.2 mmolesFe) and fluorescein labeled hen egg white avidin (7.5×10⁻⁵ mmoles)(Pierce, Rockford, Ill.) were dialyzed against 0.01M sodium acetatebuffer, pH 6.0 for 2 hours. Sodium periodate (46 μmol) was added to theavidin, incubated for 30 min at room temperature in the dark, anddialyzed against 150 mM sodium chloride. The oxidized avidin was addedto amino-CLIO, and the pH adjusted by the addition of 100 μl of 0.2 Msodium bicarbonate, pH 9.5. The mixture was incubated for 2.5 hours withstirring. Sodium cyanoborohydride was added (80 μmol) and the mixturewas incubated for 3.5 hours at room temperature. The avidin-CLIOnanoparticle was separated from unreacted avidin using a magneticseparation column (Miltenyi Biotec, Auburn, Calif.). Iron was determinedspectrophotometrically, and protein by the BCA method (Pierce). Thenumber of avidins attached per nanoparticle was calculated using amolecular weight of 67 kDa for avidin and 2064 Fe atoms per crystal forCLIO.

FIG. 10b shows that in the absence of caspase or in the presence ofcaspase inhibitor, the binding moiety is intact and therefore, so is theaggregate. When the inhibitor is not added or capase is added, theaggregate is dispersed resulting in an increase of T2. These experimentsindicate that the assay system has the sensitivity to detect enzymeactivity in solution.

Example 9: Assay for Protein (GFP) Using a Biotinylated PolyclonalAnti-GFP

Avidin-CLIO nanoparticles made as described above were reacted withbiotinylated polyclonal anti-GFP (Research Diagnostics Inc.) and werethen attached to the particles. Unreacted molecules were removed. Toprobe for protein-protein interactions, GFP (33 pmol, 200 nM) wasincubated with anti-GFP-CLIO (10 μg Fe/ml) and T2 relaxation times wererecorded (FIG. 10a ).

The results show that the presence of the antibodies resulted in adecrease of T2, indicating that the antibody conjugates were bound tothe antigen target resulting in aggregate formation.

Example 10: Assay for Caspase Using a Dibiotinylated Peptide

In this assay, a dibiotinylated peptide is synthesized that includesbetween the two biotins a “substrate” (cleavage site) that can becleaved by the protease caspase. An example of a biotinylated peptidethat is convenient to make has the general structurebiotin-G-X1-X2-X3-X4-G-K-biotin, wherein X1 to X4 are residues providingprotease specificity (the cleavage site was between X1 and X4).FmocK(Dde) can be used in the synthesis of the peptide. It can bedeprotected with 2% hydrazine and biotin coupled to the epsilon aminogroup of lysine. Alternatively, a biotinylated form of lysine can bepurchased. Biotin is added to the N terminus of the peptide through thecarboxyl group of biotin using HOBT/HBTU as activating/coupling agents.

To perform the assay, the dibiotinylated peptide is incubated with theprotease and cleavage allowed to occur. Avidin-CLIO, made as describedherein, is then added. When mixed with dibiotinylated peptide, smallaggregates form (if no protease is present). If the dibiotinylatedpeptide was cleaved, by a protease such as caspase, the monobiotinylatedproducts bind to the avidin-CLIO, but do not induce the formation ofsmall aggregates.

Example 11: Assay for Endonuclease Using Aggregates of Double-StrandedOligonucleotides

Two self-complementary 3′-alkanethiololigonucleotides:AAT-GCG-GGATCC-TAC-GAG-(CH₂)₃-SH (SEQ ID NO:16) andCTC-CTA-GGATC-CGC-ATT-(CH₂)₃-SH (SEQ ID NO:17) were conjugated tonanoparticles as described in Example 3. The resulting conjugates(Magnetic Relaxation Switches, MRS), denoted P1(AAT-GCG-GGATCC-TAC-GAG-(CH₂)₃-S-S-CLIO) and P2(CTC-CTA-GGATC-CGC-ATT-(CH₂)₃-S-S-CLIO) have on average 3oligonucleotides per particle. The formation of MRS aggregates uponmixing P1 and P2 was determined by atomic force microscopy (AFM)(Dimension 3100, Digital Instruments). Images were recorded usingtapping mode and a surface area of 5×5 μm.

The restriction endonuclease digestion was performed at 37° C. with 0.4U/μl of BamHI, (New England BioLabs) in 500 μl of 10 mM Tris HCl, 10 mMMgCl₂, 50 mM NaCl pH 7.4 containing the MRS aggregate P1/P2 (10 μg ofFe/mL). The water relaxation of the solution was measured at either timeintervals or after a one-hour incubation and compared to control samples(with no enzyme) using a 0.47 T NMR relaxometer (Bruker NMR Minispec,Billerica, Mass.).

The oligonucleotide sequences were chosen so that P1 and P2 wouldself-hybridize with the formation of a MRS aggregate that exhibits amore pronounce effect on T2. A pair of MRS (P1 and P2) thatself-assemble to form a BamHI recognition site (FIG. 1a ) was prepared.P1 (10 μg Fe/mL) had a T2 of 61.6±0.3 msec, while P2 (10 μs Fe/mL) had aT2 of 60.4±0.5 msec. Meanwhile, the T2 of an equimolar mixture of P1 andP2 (total 10 μs Fe/mL) had a T2 of 32.3±0.6 msec (p<0.0001), due toself-hybridization of the particles and formation of aggregates.Incubation with BamHI, resulted in an increase in T2 back to baselinelevels (59.4±0.4 msec). T2 changes were specifically inhibited by theaddition of a synthetic complementary oligonucleotide and otherendonucleases did not cause an increase in T2. Atomic force microscopyrevealed that P1/P2 consisted of stable aggregates with average sizesraging from 300 to 400 nm (FIG. 12a ). After a one-hour incubation withBamHI, the aggregates were no longer present and monodispersenanoparticle conjugates (50-60 nm) were observed instead (FIG. 12b ).

Example 12: Protein Assay Using Monoclonal Antibody-NanoparticleConjugates

Monoclonal antibodies can be coupled to polymer coated magneticnanoparticles using a variety of chemistries (see, e.g., Weissleder etal., U.S. Pat. No. 5,492,814; and Kang et al. (2002) BioconjugateChemistry, 13, 122-127). A useful method is that of Kang because it usesthe amino-CLIO chemistry described above and avoids destroying thedextran with oxidative treatments with periodate.

In this assay format a P1 (first monoclonal attached to a nanoparticle)and P2 (second monoclonal attached to a nanoparticle) are synthesized inseparate reactions. The target protein must contain epitopes for bothmonoclonals, so that small aggregates can form in solution. If a targetantigen is in solution, the monoclonal antibodies will bind bothepitopes on the antigen, thereby aggregating the nanoparticles,resulting in a decrease of T2.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An aggregate comprising a plurality of conjugates, wherein eachconjugate comprises a magnetic nanoparticle linked to a binding moietythat specifically binds to a target molecule, to another binding moiety,or to an aggregation inducing molecule, and wherein each conjugatewithin the aggregate is bound to at least one other conjugate in theaggregate through their respective binding moieties.
 2. The aggregate ofclaim 1, wherein the aggregate comprises 2 to 20 conjugates.
 3. Theaggregate of claim 1, wherein the aggregate has a size of about 100 to500 nm. 4-35. (canceled)
 36. A conjugate comprising a magneticnanoparticle linked to a first binding moiety, wherein the first bindingmoiety comprises a cleavage site for a target molecule and specificallybinds to an aggregation inducing molecule, forms a cleavage site for thetarget molecule when the first binding moiety binds to a second bindingmoiety, or specifically binds to an aggregation inducing molecule thatcomprises a cleavage site.
 37. The conjugate of claim 36, wherein thefirst binding moiety comprises a polypeptide that comprises the cleavagesite, and wherein the target molecule is an enzyme.
 38. The conjugate ofclaim 36, wherein the first binding moiety binds to a second bindingmoiety to form the cleavage site that is selectively cleaved by a targetmolecule, and wherein the target molecule is an enzyme. 39-73.(canceled)
 74. A method for purifying a target molecule from a sample,the method comprising obtaining a conjugate comprising a nanoparticlecomprising a magnetic metal oxide linked by a cleavable bond to abinding moiety that specifically binds to a binding site on the targetmolecule; obtaining a sample containing the target molecule in a fluid;mixing the conjugates with the sample under conditions sufficient toenable target molecules in the sample to bind to the binding moiety onthe conjugate to form target molecule-binding moiety complexes;separating the conjugates from the sample; and cleaving the cleavablebond to separate the target molecule-binding moiety complexes from theconjugates, thereby purifying the target molecules.
 75. The method ofclaim 74, wherein the target molecule is a nucleic acid, and the bindingmoiety is an oligonucleotide that is complementary to a portion of thetarget nucleic acid.
 76. The method of claim 74, wherein the targetmolecule is a polypeptide, and the binding moiety is an antibody thatspecifically binds to a portion of the target polypeptide. 77-82.(canceled)